Plant glutamate receptors

ABSTRACT

The present invention relates to a family of GluR in plants, including ionotropic (iGluR), metabotropic (mGluR) and other glutamate-like plant receptors. The plant GluRs of the invention may function as signal transducers involved in the regulation of plant growth. The invention also relates to the identification of compounds that modulate the activity of the plant GluR, and the use of such compounds as plant growth regulators, including herbicides.

This is a division of application Ser. No. 08/658,335, filed Jun. 6,1996 now U.S. Pat. No. 5,981,703 which is a continuation-in-part ofapplication Ser. No. 08/629,291, filed Apr. 8, 1996 now U.S. Pat. No.5,959,174, which is a continuation-in-part of application Ser. No.08/481,956, filed Jun. 7, 1995 now U.S. Pat. No. 5,824,867.

This invention was made with U.S. government support under NIH grantGM-32877. The U.S. government has certain rights in the invention.

1. INTRODUCTION

The invention relates to a family of glutamate receptors (GluR) inplants, compounds that modulate the activity of the plant GluR, and theuse of such compounds as plant growth regulators, including herbicides.The invention also relates to nucleotide sequences encoding the plantGluR and to plant assay systems designed to identify novel plant growthregulators that may be used as herbicides and/or pharmaceutical drugs.

2. BACKGROUND OF THE INVENTION 2.1. Metabolic and Regulatory Roles ofGlutamate in Plants

Glutamate has important roles in plant nitrogen metabolism. Glutamate isthe amino acid into which inorganic nitrogen is first assimilated intoorganic form. Plants have three distinct nitrogen processes related tonitrogen metabolism: (1) primary nitrogen-assimilation, (2)photorespiration, and (3) nitrogen “recycling.” All three processesinvolve assimilation of ammonia into glutamate and glutamine by theoperation of glutamine synthetase (GS) and glutamate synthase (GOGAT).Glutamate and glutamine, being the first products ofnitrogen-assimilation, in turn serve as nitrogen donors in thebiosynthesis of essentially all amino acids, nucleic acids, and othernitrogen-containing compounds such as chlorophyll (Lea et al., in:Recent Advances in Phytochemistry, edited by Poulton et al., New Yorkand London: Plenum Press, 1988, pp. 157-189).

Glutamate is also a principal “nitrogen-transport” compound in plants.It and glutamine are two major amino acids used to transport nitrogenwithin a plant (Lea and Miflin, in: The Biochemistry of Plants, Vol. 5,edited by Stumpf and Conn, Academic Press, 1980, pp. 569-607; Urquhartand Joy, 1981, Plant Physiol. 68:750-754). In light-grown metabolicallyactive plants, glutamate and glutamine are used in anabolic reactionsand are transported as such. By contrast, in etiolated or dark-adaptedplants, glutamine is converted into inert asparagine for long-termnitrogen storage.

Glutamate also may be a signal or regulatory molecule in regulating theexpression of plant genes. Specifically, glutamate along with glutamineand asparagine appears to have an antagonistic role to that of sucrosein regulating certain nitrogen assimilation genes. Sucrose has beenshown to induce the expression of genes for nitrate reductase (NR),nitrite reductase (NiR), and chloroplastic glutamine synthetase (GS2) intobacco (Saur et al., 1987, Z. Naturforsch. 42:270-278; Vincentz et al.,1993, Plant J. 3:315-324). Sucrose also induces genes for GS2 andferroredoxin-dependent glutamate synthase (Fd-GOGAT) in Arabidopsis.Sucrose-induction of the NR and NiR in tobacco is suppressed bysubsequent additions of glutamine, glutamate or asparagine to the media(Vincentz et al., ibid.). Conversely, a nitrogen metabolism gene,glutamine-dependent asparagine synthetase (ASN1), in Arabidopsis isrepressed by light or sucrose (Lam et al., 1994, Plant Physiol.106:1347-1357). The sucrose repression of ASN1 can be relieved byadditions of glutamine, glutamate, or asparagine (Id.).

2.2. Glutamate Receptors in Animal Cells

Excitatory amino acids constitute the principal neurotransmitterreceptors that mediate synaptic communication in animals (Gasic et al.,1992, Annu. Rev. Physiol. 54: 507-536). In particular, L-glutamate isthe major excitatory neurotransmitter of the mammalian central nervoussystem (Monaghan et al., 1989, Annu. Rev. Pharmacol. Toxic. 29:365-402). Glutamate signaling in animals is important for manyphysiological and pathological processes such as developmentalplasticity, long-term potentiation, and excitotoxic damage in ischemiaand other neurodegenerative disorders (Choi, 1988, Neuron 1: 623-624;Kennedy, 1989, Cell 59: 777-787).

In animals, glutamate can trigger various downstream physiologicalresponses by interacting with different GluR. GluRs in animals areinvolved in central nervous system (CNS) disorders such as Huntington'sdisease, Parkinson's disease and Alzheimer's disease. The GluR isinvolved in the initiation and propagation of seizures and in massiveneuronal cell death during periods of ischemia and hypoglycemia. GluRshave been grouped into five distinct subtypes (Gasic et al., 1992, Annu.Rev. Physiol. 54: 507-536): (a) NMDA (N-methyl-D-aspartate), (b) KA(Kainate), (c) AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate), (d) L-AP4 (2-amino-4-phosphonobutyrate) and (e) ACPD(trans-1-amino-cyclopentane-1,3 dicarboxylate). NMPA, KA and AMPA, whichform ligand gated ion channels that are activated on a msec scale, arethe ionotropic (iGluR) subtypes. By contrast, metabotropic (mGluR)subtypes, L-AP4 and ACPD, are coupled to G proteins and operate on atime scale of several hundred msec to seconds. LAP-4 receptor probablyacts via a G protein by increasing the hydrolysis of cGMP andsubsequently leads to the closure of ion channels conducting an inwardcurrent. The ACPD subtype, which couples with a G protein that is linkedto inositol phosphate/diacylglycerol formation and subsequent release ofcalcium from internal stores. Both iGluR and mGluR seem to play a rolein the activation of transcription factors, such as c-jun and c-fos(Condorelli et al., 1993, J. Neurochem. 60: 877-885; Condorelli et al.,1994, Neurochem. Res. 19: 489-499).

2.2.1. Ionotropic Glutamate Receptors

There are major differences in the neurophysiological functions of thethree subtypes of iGluR (Seeburg, 1995, TINS 16:359-365). AMPA receptorsare found in the majority of all fast excitatory neurotransmission. Thevery low Ca++ permeability of AMPA receptor suggests that they probablydo not trigger biochemical reactions directly via an increase inintracellular Ca++ levels. In NMDA receptor, Ca++ flux will triggerdifferent processes ranging from trophic developmental actions to anactivity-dependent resetting of the synaptic strength underlying someforms of learning and memory. The significance of high-affinity kainatesites in the nervous systems is yet to be fully understood.

(A) AMPA Receptor

AMPA receptors consist of at least four different subunits: GluR1-GluR4.The two major forms, named “flip” and “flop”, which are formed bydifferential splicing, display different expression profiles in themature and the developing brain (Sommer et al., 1990, Science249:1580-1585). For GluR2 subunit, RNA editing (Q to R) in transmembranedomain (TM) II has been shown to regulate the Ca++ permeability. RNAediting leads to a decrease in Ca++ permeability (Burnashev et al.,1992, Neuron, 8:189-198; Hume et al., 1991, Science 253:1028-1031).

(B) Kainate Receptors

High-affinity kainate receptors are composed of subunits GluR5-GluR7,KA1, and KA2 (Seeburg et al., 1995, TINS 16:359-365). Both GluR5 andGluR6 subunits also display the Q to R editing similar to the case ofGluR2 of AMPA receptors (Sommer et al., 1991, Cell 67:11-19). GluR6 hastwo additional positions in TMI that are modified by RNA editing (Kohleret al., 1993, Neuron 10:491-500). For GluR6, only when TMI is editeddoes editing in TMII (Q to R) influence Ca++ permeability (Kohler etal., 1993, Neuron 10:491-500). In contrast to the AMPA receptor channel,GluR6(R) channels edited in TMI show a higher Ca++ permeability thanGluR6(Q) channels (Kohler et al., 1993, Neuron 10:491-500).

(C) NMDA Receptor

NMDA receptors are highly permeable to Ca++. The NMDA receptor can bereconstituted as heteromeric structures from two subunit types: NRI andone of the four NR2 (NR2A-NR2D) (Seeburg, 1995, TINS 16:359-365). All ofthe subunits do not show RNA editing in TMI and TMII. In fact allsubunits contain an N at the site which Q to R editing occurs innon-NMDA iGluR. The most distinct feature of NMDA receptors is that theyrequire both glycine and glutamate or both glycine and NMDA to activatethe channel. The NMDA receptor has been linked to regulation ofcoccidian rhythm in rat brains.

2.2.2. Metabotropic Glutamate Receptors

In contrast to ionotropic glutamate receptors. (iGluR) the hallmark ofthe mGluR receptors resides on the fact that these molecules are coupledto G proteins and thus able to elicit typical G protein-drivenintracellular responses (Gasic et al., 1992, Annu. Rev. Physiol.54:507-536; Minakami et al., 1994, Biochem. Biophys.. Res. Commun.199:1136-1143; Schoepp et al., 1993, Trends in Pharmacol. Sci.14:13-20).

The cloning of mGluR1 from a expression cDNA library of a rat cerebellum(mGluR1a), was followed by the cloning and characterization of six othermGluR genes (Schoepp et al., 1993, Trends in Pharmacol. Sci. 14:13-20).The mGluR1 a, the prototype member of the family, possess a largeextracellular domain, a putative “seven pass” transmembrane region anddisplay highly conserved amino acids with other members of the mGluRsboth at the membrane spanning region, extracellular region andintracytoplasmic loops between transmembrane domains (Gasic et al.,1992, Annu. Rev. Physiol. 54:507-536; Minakami et al., 1994, Biochem.Biophys. Res. Commun. 199:1136-1143; Schoepp et al., 1993, Trends inPharmacol. Sci. 14:13-20). The mGluR genes are unique in that they donot show significant homology with any of the previously characterized Gproteins (Nakanishi et al., 1994, In: Toward a molecular basis ofalcohol use and abuse. ed. by Jansson et al. p 71-80; Schoepp et al.,1993, Trends in Pharmacol. Sci. 14:13-20) and very little is known onthe signal transduction mechanisms and second messenger responses foreach mGluR receptor (Schoepp et al., 1993, Trends in Pharmac. Sci.14:13-20).

Studies in the in situ localization of mRNA encoding the differentmGluRs shows them to be differentially distributed in the brain withcells from diverse tissues expressing one or more combinations of thevarious members of the mGluR family of receptors, suggestive of arelevant participation in the modulation of several important biologicalprocesses (Schoepp et al., 1993, Trends in Pharmac. Sci. 14:13-20).Indeed, the mGluR proteins have been reported to be involved withneuroprotection and neuronal pathophysiology (Baskys, 1992, Trends inNeuro Sci. 15:92-96; Schoepp et al., 1993, Trends in Pharmac. Sci.14:13-20).

Analysis of the pharmacological properties of the individual mGluRmolecules revealed that the agonists L-AP4(L-2-amino-4-phosphonobutyrate) and ACPD (trans-1-amino-cyclopentane-1,3dicarboxylate) can selectively stimulate different mGluR serving as abasis for the classification of this group of proteins (Nakanishi etal., 1994, In: Toward a molecular basis of alcohol use and abuse. Ed.Jansson et al. p 71-80).

(A) L-AP4 Receptor

The L-AP4 receptor has been defined electrophisologically as aninhibitory glutamate site and biochemical evidence suggest that mGluR4,mGluR6 and mGluR7 are involved in this response (Nakanishi et al., 1994,In: Toward a molecular basis of alcohol use and abuse ed by Jansson etal. p 71-80; Schoepp et al., 1993, Trends in Pharmacol. Sci. 14:13-20).The L-AP4 receptor appears to be localized pre-synaptically such thatactivation inhibits the release of excitatory neurotransmitter through amechanism involving a pertussis toxin sensitive G protein (Nakanishi etal., 1994, In: Toward a molecular basis of alcohol use and abuse. Ed.Jansson et al. p 71-80). The molecular identity and possible biologicalfunction of this group of receptors come from studies where mammaliancells were transfected with a cloned isoform of mGluR (mGluR4) respondedto both L-glutamate and L-AP4 by depressing forskolin-stimulated cAMPlevels (Nakanishi et al., 1994, In: Toward a molecular basis of alcoholuse and abuse. ed. by Jansson, p 71-80; Schoepp et al., 1993, Trends inPharmacol. Sci. 14:13-20). L-AP-4 has also been shown to reduceelectrically-stimulated excitatory transmission, suggestive of a closeinteraction between mGluR and Ca²⁺ channels (Cunningham et al., 1993,Life Sciences 54:135-148; Schoepp et al., 1993, Trends in Pharmac. Sci.14:13-20) and specifically in the regulation of ionotropic glutamatereceptors (Baskys, 1992, Trends in Neuro. Sci. 15:92-96). Very little isknown about the signal transduction mechanisms and second messengerresponses elicited by this subgroup of mGluR.

(B) ACPD Receptor

The mechanisms of signal transduction of this subgroup of receptors isbetter understood than that of the L-AP4-responsive mGluRs. Transfectionof the cDNA for mGluR1, mGluR2, mGluR3 and mGluR5 in CHO cells revealedthat this receptors are strongly responsive to the drug ACPD(trans-1-amino-cyclopentane-1,3 dicarboxylate) (Cunningham et al., 1993,Life Sciences 54:135-148; Schoepp et al., 1993, Trends in Pharmac. Sci.14:13-20). However, not all mGluR activate the same pathways anddifferent mGluR can elicit diverse intracellular responses. Thus, mGluR5possess high homology with mGluR1 yet these two receptors differ inwhich mGluR5 does not induce formation of cAMP. Moreover, stimulation ofthe mGluR2 and 3 does not lead to phosphoinositide hydrolysis and mGluR2has been shown to inhibit cAMP formation in transfection experiments(Schoepp et al. 1993). The diversity of the mGluR family of receptorscan be further appreciated by the recent observation that mGluR2 andmGluR3 receptors display an unusually high response to stimulation byquisqualate when compared to other ACPD mGluR responses (Nakanishi etal., 1994, In: Toward a molecular basis of alcohol use and abuse. ed. byJansson et al. p 71-80; Schoepp et al., 1993, Trends in Pharmacol. Sci.14:13-20) which argues in favor of their further grouping in a morespecialized division among the ACPD-induced receptors. Finally,stimulation of primary neuronal cultures with ACPD and quisqualatecaused a strong and transient induction of immediate early genes such asc-fos, c-jun and zif-268 mRNAs (Condorelli et al., 1994, Neurochem Res.19:489-499).

3. SUMMARY OF THE INVENTION

The present invention relates to a family of GluR in plants, includingionotropic (iGluR), metabotropic (mGluR) and other glutamate-like plantreceptors. The plant GluRs of the invention may function as signaltransducers involved in the regulation of plant growth. The inventionalso relates to the identification of compounds that modulate theactivity of the plant GluR, and the use of such compounds as plantgrowth regulators, including herbicides.

The invention is based in part, on a number of unanticipated surprisingdiscoveries. One is the discovery of plant proteins that have highdegree of amino acid sequence homology to the animal ionotropic ormetabotropic glutamate receptors previously found only in vertebratetissues. The other is the finding that agonists and antagonists ofanimal glutamate receptors function to modulate expression of plantgenes and as plant growth regulators. These agonists and antagonistsstructurally constrained or do not resemble glutamate. Thus, theiractions in plants likely are due to their specific interaction with oneor more plant glutamate receptors, rather than to general effects onglutamate-utilizing enzymes. These findings together indicate thatplants have glutamate receptors that function as signal transducers.

The invention encompasses: (a) nucleotide sequences that encode theplant GluR, including mutants, recombinants, and fusion proteins; (b)the expression of such nucleotide sequences in genetically engineeredhost cells and/or in transgenic plants; (c) the isolated GluR plantproteins and GluR engineered gene products, including mutants,fragments, and fusion proteins; (d) antibodies to the plant GluRproteins and polypeptides; (e) screening assays involving the use ofplants, transgenic plants, genetically engineered cells that express theplant GluR or mutants thereof, or GluR proteins or peptides, to identifycompounds that act as agonists or antagonists; (f) the use of suchagonists or antagonists as plant growth regulators, includingherbicides; (g) the engineering of transgenic plants resistant toherbicidal antagonists of the plant GluR and/or transgenic plants withimproved agronomic or industrial properties; and (h) the use ofantagonists or agonists of the plant GluR identified in the screeningassays described herein as drugs for animal use, including humans.

3.1. Definitions

An agonist is defined herein as an agent that acts like a referencedcompound or that activates a receptor molecule.

An antagonist is defined herein as an agent that acts in opposition toan agonist or a referenced compound or that inhibits a receptormolecule.

A chimeric gene comprises a coding sequence linked to a regulatoryregion, i.e., promoters, enhancer elements and additional elements knownto those skilled in the art that drive and regulate expression, thatsaid coding sequence is not naturally linked to. The coding sequence mayencode messenger RNA (mRNA), antisense RNA or ribozymes.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A,1B,1C. HPLC Analysis of Free Amino Acids in Arabidopsis

FIG. 1A. Amino acids were extracted from leaves of Arabidopsis plantsthat were grown in light (empty boxes) or subsequently dark adapted for24 hours (filled boxes). Amino acids were derivatized and separated byreverse phase HPLC. Each sample represents the average of threedifferent plants (two leaves/plant). The standard three letter code isused for all amino acids; gaba: γ-amino burytic acid.

FIG. 1B. Average amino acid content in phloem exudates of threeindependent plants (one leaf/plant).

FIG. 1C. Average amino acid content of xylem sap collected from cuthypocotyls of three independent plants. Data are from Schultz (1994).

FIG. 2. Reciprocal Control By Light On Arabidopsis GLN2 and ASN1Expression

The effects of light on GLN2 and ASN1 expression were tested in matureArabidopsis plants. Plants were grown on soil under a 16-h light/8-hdark cycle for 2 weeks and transferred to continuous light (lane 1) orcontinuous darkness (lane 2) for 5 d. Total RNA (10 μg) was used foreach of the lanes. Hybridization was performed by [α-³²P]dATP-labeledGLN2 or digoxigenin-labeled ASN1 DNA probes in Strategene QuikHybsolution under high stringency condition. The nylon filter was firsthybridized with the GLN2 probe, then stripped, and rehybridized with theASN1 probe. (Lam et al., 1994).

FIG. 3. Effect Of C:N Ratio On The mRNA Levels Of ASN1 And GDH

Arabidopsis seeds were grown on plates containing MS medium plus 3%(w/v) Suc under 16-h light/8-h dark cycle for 2 weeks. The plants werethen transferred to media described below and grown in complete darknessfor 2.5 d. Lanes 1 to 4, MS medium with no sugar; lanes 5 to 8, MSmedium with 3% (w/v) Suc. MS was supplemented with 0.4 mM Asn (lanes 2and 6), 3.4 mM Gln (lanes 3 and 7), or 3.3 mM Glu (lanes 4 and 8). Theexpression of ASN1, GDH, and a cytosolic GS (GSR2) were detected bynorthern analyses (under high-stringency conditions in 50% [v/v]formamide solution). 10 μg of total RNA was used for each lane. Thenylon filter was first hybridized with ASN1, then stripped, andre-hybridized with the GSR2 probe. The nylon filter was then strippedagain and re-hybridized with the GDH probe.

FIGS. 4A and 4B. A Model Depicting The Regulation Of NitrogenAssimilation Genes By C:N Ratio

FIG. 4A. In the light, when photosynthesis occurs and carbon skeletonsare abundant, nitrogen is assimilated and transported as glutamine andglutamate; levels of mRNA for genes involved in glutamine and glutamatesynthesis (GLN2, GLU1) are accordingly induced by both light andsucrose. By contrast, light represses the synthesis of asparagine whichtherefore accumulates only in tissues of dark-adapted plants.

FIG. 4B. Levels of ASN1 mRNA are dramatically induced in dark-adaptedplants, and this induction is repressed by light or by high levels ofsucrose. Thus, under conditions of carbon limitation or nitrogen excess,plants activate genes for asparagine biosynthesis (Lam et al., 1995).The mRNA level of GDH was found to be under similar control (see alsoFIG. 3).

FIG. 5. Proposed Topology and Functional Domains of Ionotropic GlutamateReceptor Subunits

Hydrophobicity plots of GluR subunit sequences predict fourtransmembrane (TM) segments (TM I-IV), depicted here hypothetically as ahelices I-IV.

FIG. 6. Peptide Sequence Homology Between The Arabidopsis iGluR (SEQ IDNO1) and Animal iGluRs [E. coli GlnH:(SEQ ID NO:2); Chick KBP:(SEQ IDNO:3); Frog KBP;(SEQ ID NO:4); Rat GluR-K1: (SEQ ID NO:5); RatGluR-K3:(SEQ ID NO:6); Rat GluR-K2:(SEQ ID NO:7)]

Peptide sequence analysis shows that the putative Arabidopsis iGluRcontains a conserved glutamine binding domain which exists in all animaliGluRs.

FIG. 7A. Peptide sequence analysis shows the extensive homology betweenthe putative Arabidopsis iGluR (SEQ ID NO:8) animal iGluRs. The regionof homology extends from the glutamine binding domain into thetransmembrane domains.

FIG. 7B. Peptide sequence analysis shows the extensive homology betweenthe putative Arabidopsis mGluR and animal iGluR [NMDA:(SEQ ID NO:9);KA:(SEQ ID NO:10)]. The region of homology extends from the glutamatebinding domain into the transmembrane domain.

FIG. 7C. Arabidopsis EST clones with low degree homology to glutamatebinding domains. These EST clones have no homology to ionotropic normetabotrobic GluR. Partial nucleotide sequence of EST clones areprovided here [ATT50711:(SEQ ID NO:13); ATT52655:(SEQ ID NO:14;T20773:(SEQ ID NO:15)].

FIG. 8. Genomic Southern Analysis of Arabidopsis iGluR

Two μg of CsCl-purified Arabidopsis genomic DNA was digested withdifferent restriction enzymes. Genomic Southern blot analyses wereperformed by running the digested DNA on a 1% (w/v) Tris-phosphate-EDTAagarose gel. The DNA was transferred to a nylon membrane afterdepurination, denaturation, and neutralization steps, followed byhigh-stringency hybridization with DIG-labeled probes which aregenerated by random-primed reactions (as described in theBoehringer-Mannheim Genius System User's Guide).

FIG. 9. Expression Of Arabidopsis iGluR In Different Tissues

Twenty μg of total RNA from each of the leaf, root, and flower tissueswere run on a 1% formaldehyde agarose gel. Northern blot analyses wereperformed with high-stringency hybridization conditions at a temperatureof 42° C. in 50% (v/v) formamide hybridization solution. Washing andchemiluminescent detection were performed according to theBoehringer-Mannheim Genius System User's Guide. The Northern shows thatArabidopsis iGluR mRNA is expressed predominantly in leaves and also atlower levels in roots and flowers of Arabidopsis.

FIG. 10. Chemical Structures of Glutamate, Kainate, and DNQX

FIGS. 11A and 11B. Effects Of iGluR Agonist and On The Growth OfArabidopsis

Arabidopsis seeds were grown on MS+3% sucrose vertical tissue cultureplates containing various amounts of kainate (FIG. 11A) or DNQX (FIG.11B), with (white bars) or without (black bars) glutamatesupplementation. The effects of each drug on plant growth were assayedby measuring root length after two week. The results were discussed intext.

FIGS. 12A, 12B, 12C. Induction of Gene Expression by iGluR Agonist

FIG. 12A. Arabidopsis seeds were grown on plates containing MS mediumplates 3% (w/v) Suc under 16-h light/8-h dark cycle for 2-3 weeks. Theplants were then transferred to media described below and grown incomplete darkness for 2 d. All samples containing 3% sucrose except forlane 2. MS was supplemented with kainate (lane 4:0.3 mM and lane 5:0.03mM), 0.05% (w/v) glutamate (lanes 6 and 7), and 10 μM DNQX (lanes 7 and8). The expression of ASN1 and a control gene were detected by northernanalyses on duplicate blots (under high-stringency conditions in 50%[v/v] formamide-solution), 20 μg of total RNA was used for each lane.

FIG. 12B. Quantitation of the Northern blot results in (A) bydensitometry scan.

FIG. 12C. Average folds of induction in two Northern lot experiments.

FIGS. 13A and 13B. Inhibition of Arabidopsis Growth by High Dosage ofKainate and DNQX

Photographic representation of high dosage inhibitory effects of kainateand DNQX on the growth of Arabidopsis as described in FIG. 11.

FIGS. 14A and 14B. Model Depicting Effects of Agonists and Antagonistson Plants Expressing Wildtype and Mutant GluR.

FIG. 15. Nucleotide and deduced amino acid sequence of full lengthArabidopsis iGluR cDNA, called iGlr1. The regions of highest homology toanimal iGluR are denoted in FIGS. 17 and 18.

The full-length Arabidopsis iGlr1 cDNA clone was constructed as follows:the partial EST cDNA clone 107M14T7 was used as a hybridization probe toisolate two additional iGlr cDNA clones (HM299 and HM262) from twodifferent Arabidopsis cDNA libraries, KC-HM1 and CD4-7 (obtained fromthe Arabidopsis stock center, Ohio). Portions of each iGlr cDNA clonewere annealed to generate a full-length Arabidopsis iGlr1 cDNA which wasgiven the trivial name HM330.

FIG. 16. Proposed membrane topology of iGluR receptors in animals. Themodel shows the important domains of animal iGluRs and their membranetopology. This figure is included as a reference, and does not includedata generated in our lab. In addition to a signature 3+1 transmembranetopology, animal iGluRs contain two extracellular domains which areproposed to bind to glutamate. The two putative glutamate bindingsdomains have been previously shown to have homology to the E. coliglutamine permease gene (GlnH).

FIG. 17. Conserved domains between animal and Arabidopsis iGluR gene.The Arabidopsis iGlr1 cDNA encodes numerous conserved features of animaliGluRs including, 1. a signal peptide to direct it to the membrane (SP),2. two putative glutamate-binding domains with homology to E. coli(GlnH1 and GlnH2), 3. Four transmembrane domains (TM I-IV). The aminoacid sequences spanning the high homology region are shown in FIG. 4.

FIG. 18. Amino acid identities between Arabidopsis iGlr1 (SEQ IDNOS:26-27) and iGluR (SEQ ID NOS:28-31) gene of rat. Boxed are the GlnH1and GlnH2 domains which show homology to E. coli glutamine permease (asdefined in the animal sequence; see FIG. 17), the four transmembranedomains (TM I-IV). The arrow points to the conserved ligand-bindingresidue in the GlnH1 domain.

FIG. 19. A proposed role for plant iGluR in light signal transduction.We have shown that the iGluR antagonist DNQX and high concentrations ofthe iGluR agonist KA can block photomorphogenic processes such asgermination, chloroplast development and hypocotyl elongation (see FIGS.20-23). This model proposes a role for plant iGluR in the light signaltransduction cascade.

FIG. 20. iGluR antagonist DNQX phenocopies Arabidopsis long hypocotyl(hy) mutants impaired in light signal transduction. Light normallypromotes greening and inhibits hypocotyl elongation in wild-typeseedlings. hy mutants are impaired in light perception/signaltransduction. hy mutants when grown in light take on the morphology ofdark-grown seedlings (long hypocotyl). When wild-type plants are treatedwith DNQX, they grow as hy mutants (long hypocotyl).

FIG. 21. DNQX has a significant effect on hypocotyl length inArabidopsis. Increasing doses of DNQX (200 uM and 400 uM) causesignificant increases in hypocotyl elongation in Arabidopsis. N=numberof plants measured.

FIG. 22. DNQX, the iGluR antagonist blocks light-induced chloroplastdevelopment in Arabidopsis. Left panel. Plants grown in darkness haveunopened yellow cotyledons. When these plants are exposed to light for 5hrs, the cotyledons begin to green. If plants are grown in the dark withDNQX in the media, the cotyledons remain yellow and unopened after 5 hrsof light exposure. Thus, DNQX appears to block light-induced chloroplastdevelopment.

FIG. 23. Effects of kainate of germination of Arabidopsis in the dark.Arabidopsis seedlings germinated on media containing increasing amountsof kainate (200-400 uM) show a significant inhibition of germination ofdark-grown seedlings. This inhibition of germination is likely to bespecific to iGluR as it is specifically reversed by the supplementationof glutamate to the growth media.

FIG. 24. The Arabidopsis iGlr1 gene was mapped using recombinant inbredlines of Arabidopsis. An RFLP for iGlr1 was identified in the wild-typeArabidopsis ecotypes Columbia (C) and Landsberg (L). This iGlr1-specificRFLP was used to identify the genotype of the iGlr1 gene in 30Recombinant Inbred lines as being derived from the C or L parents. The“pattern” of inheritance of the iGlr1 gene in the recombinant inbredlines was compared to known markers and used to determine a map position(see FIG. 25).

FIG. 25. iGlr1 maps to chromosome III to a similar position as two knownmutants, hy2 and spy. Using data from recombinant inbred lines hy2 is amutant impaired in light signal transduction (see review Whitelam &Harberd, Plant Cell Environment 1994, 17, 615-625). The spy mutant isimpaired in GA hormone signal transduction (Jacobsen & Olszewski, 1993,Plant Cell 5, 887-896).

FIG. 26. Screen for Arabidopsis mutants with altered sensitivity to theiGluR antagonist DNQX. Mutants affected in iGluR can be used to test thein vivo function of plant iGluR and could also be mapped relative to thecloned gene. To isolate mutants in Arabidopsis iGluR we developed ascreen for plants that are super-sensitive to the iGluR antagonist DNQX.Normally wild-type Arabidopsis only show an elongated hypocotylphenotype when exposed to high doses of DNQX (200-400 μM) and show nohypocotyl elongation at low-concentrations (100 μM) (see FIG. 21).Therefore, ems mutagenized M2 seeds were germinated on media containing100 uM DNQX and look for mutants that displayed an elongated hypocotylat this low dose of DNQX.

FIGS. 27A-D. Isolation of putative Arabidopsis mutants that aresuper-sensitive to the iGluR antagonist DNQX. Arabidopsis wild-typeseedlings show no significant hypocotyl elongation when germinated on100 uM DNQX (FIG. 27B) compared to control MS (FIG. 27A). By contrast,the putative DNQX supersensitive mutant shows an elongated hypocotylwhen germinated on 100 uM DNQX (FIG. 27D). The putative super-sensitivemutant also shows an elongated hypocotyl compared to wild-type whengerminated in the absence of DNQX (FIG. 27C). This is not unexpectedthat a mutation in iGluR would cause a phenotype of light-insensitivity.

FIGS. 28A and 28B. Selection of Arabidopsis mutants resistant to theiGluR agonist Kainate. High doses of kainate (12 mM) kill wild-typeseedlings. This is not unexpected as high doses of the iGluR agonistkainate function as a neurotoxin in animals. Arabidopsis mutants withputative defects in the KA binding site of iGluR were selected for theability to grow in the presence of 12 mM kainate. FIG. 28A shows emsmutagenized Arabidopsis M2 seedlings sown on 12 mM kainate. Note the oneKA-resistant plant is enlarged in FIG. 28B.

FIG. 29. Arabidopsis mutants resistant to the iGluR agonist kainate,display a Giant phenotype. Three independent Arabidopsis were mutantsselected for growth on 12 mM KA (see FIG. 28). When the putativeKA-resistant plants are transferred to soil, they each display varyingdegrees of a Giant vegetative phenotype. This result may indicate thatiGluR affects/enhances overall plant growth.

FIG. 30. Hydropathy plots of plant vs. animal iGluR. The hydropathy plotand prediction of transmembrane regions based on deduced primarysequence of rat iGluR K2 (upper panel) and Arabidopsis iGlr1 (lowerpanel) were performed with the program TMpred (K. Hofmann and W.Stoffel, TMbase—A database of membrane spanning proteins segments, Biol.Chem. Hoppe-Seyler 347,166 (1993)). Results showed that in both animaland plant iGluRs, there is a N-terminal signal peptide as well as a 3+1transmembrane domain at the C-terminal region. These are structuralfeatures conserved in all iGluR genes.

FIG. 31. Inhibitory effects of iGluR antagonist DNOX on light-inducedchlorophyll synthesis in Arabidopsis. Seedlings were sown on MS+3%sucrose agar plates and grown in complete darkness (etiolated) for 5days. On day 6, half of the seedlings were kept in the dark (D) and theother half were transferred to white light (L) for 7.5 hours. Levels ofchlorphyll a and chlorophyll b were measured using the method by Moran(Plant Physiol. 1982, 69:1376-1381). The cotyledons of 30-40 plants wereused for each data point. The results of each treatment were an averageof three groups of plants. The error bars represent standard deviation.Black bar=no DNQX treatment. White bar=treatment with 0.4 mM DNQX.Treatment with DNQX resulted in a 30-35% reduction in light-inducedchlorophyll synthesis.

FIG. 32. Late flowering in KA-resistant “giant” mutants. Two independentKA-resistant mutants (KA-giant-1 and KA-Giant-2) show a giant, lateflowering phenotype. Some of the cauline leaves from the floweringstalks of these KA-resistant giant mutants (panel B) display themorphology of rosette leaves in constrast to the normal morphology inwild type plants (panel A). These results suggest that iGluR may beinvolved in flowering and developmental processes.

FIG. 33. Isolation of a homeotic mutant from the DNQX supersensitivescreen. One DNXQ supersentitive mutant (0.1DNQX-10) showed a homeoticphenotype. Lateral roots emerged from the elongation hypocotyl of theplant (shown by the arrows). In wild type plants, lateral roots emergeonly from roots.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a family of plant glutamate receptors,and glutamate-like receptors, the identification of compounds thatmodulate the activity of the plant GluR, and their use as plant growthregulators, including herbicides, or the identification ofpharmaceutical agents used in animals, including man. The presentinvention is based, in part, on the discovery that glutamate may servenot only to transport nitrogen within a plant, but also act as asignaling molecule. A number of observations and discoveries describedherein support the conclusion that the amino acids function as signalingmolecules in plants. First, out of the 20 amino acids, only the amideamino acids, glutamate, glutamine, aspartate, and asparagine, accumulateto any significant levels as free amino acids in plant tissues,accounting for 64% of the total free amino acids in Arabidopsis leaves(FIG. 1). Second, out of the 20 amino acids only these four amide aminoacids are found circulating within the plant vasculature to significantlevels. Glutamate is the predominant amino acid transported within thephloem of light grown plants (FIG. 1 open bars). That glutamate mayserve as a signaling molecule in plants is further supported by the factthat levels of free asparagine, glutamate, glutamine and aspartate arenot static, but modulated by light. Glutamate levels are higher in thedark and low in the light (FIG. 1). In addition, the present inventionis based on the discovery that glutamate, glutamine or asparagine eachaffects the expression of several nitrogen assimilatory genes inArabidopsis (Lam et al., 1994, Plant Physiol. 106: 1347-1357; Vincentzet al., 1993, Plant J. 3:315-324). These amino acids have also beenshown to affect the expression of genes involved in nitrate reduction toammonia (Vincentz et al., 1993, The Plant Journal 3: 315-324).

The proposed role for glutamate is supported by the Applicants'identification of a full-length Arabidopsis cDNA, Glrl, that encodes aputative glutamate receptor most homologous to the iGluR class of animalglutamate receptors. The encoded GLrl protein contains all thecharacteristic features of ionotropic glutamate receptors (iGluR)including a signal peptide, two halves of the putative glutamate-bindingdomain and the three plus one transmembrane domains. Applicants haveidentified a family of iGluR genes in a variety of plants includingArabidopsis, dicots, legumes and monocots.

The proposed role for glutamate is supported by the identification oftwo Arabidopsis cDNAs with striking identity to iGluR and mGluR foundpreviously only in the animal nervous system. The Applicants identifiedtwo cDNA clones each with identity to a distinct type of animalglutamate receptor. One Arabidopsis cDNA clone (EST#107M14T7, pAt-iGR-1)shares high identity to a class of glutamate receptors called ionotropic(iGluR) receptors which constitutes ligand-gated ion channels (Gasic, G.P. and Hollmann, 1992, Annu. Rev. Physiol. 54:507-536; Kanai et al.,1993, TINS 16:365-370; O'Hara et al., 1993, Neuron 11:41-52; Seeburg,1993, TINS 16:359-365). In animals, binding of glutamate to membranebound iGluR stimulates the influx of Ca++ resulting in and fastexcitatory neurotransmission which will subsequently cause a widevariety of downstream responses (Gasic and Hollmann, 1992, Annu. Rev.Physiol. 54:507-536; Kanai et al., 1993, TINS 16:365-370; O'Hara et al.,1993, Neuron 11:41-52; Seeburg, 1993, TINS 16:359-365). For example,iGluRs may play a role in the activation of transcription factors suchas c-fos and c-jun in primary neuronal cultures (Condorelli et al.,1993, J. Neurochem. 60:877-885; Condorelli et al., 1994, Neurochem. Res.19:489-499).

The invention also relates to a second Arabidopsis cDNA (EST# 97C23T7,pAT-mGR-1) which shares identity to another class of animal glutamatereceptors called metabotropic glutamate receptor, mGluR (Gasic andHollmann, 1992, Annu. Rev. Physiol. 54:507-536). In animals, the mGluRclass of glutamate receptors is coupled to a G protein that is linked toinositol phosphate/diacylglycerol formation which results in subsequentrelease of calcium from internal stores (Gasic, and Hollmann, 1992,Annu. Rev. Physiol. 54:507-536). In animals, mGluRs also have beenreported to activate immediate early response genes, such as c-fos,c-jun, zif-268 (Condorelli et al., ibid).

The present invention is also based on the Applicants' discovery that inaddition to possessing GluR genes, plants possess functional GluRs. TheArabidopsis iGluR cDNA (pAt-iGR-1) shows high identity to the animalGluR specifically activated by an iGluR agonist called kainic acid (KA).The plant iGluR is unique in that it has homology to both NMDA-type andKA-type GluR in animals. These kainate-selective iGluR receptors arecompetitively inhibited by an iGluR antagonist called6,7-dinitroquinoxaline (DNQX). This iGluR agonist (KA) and antagonist(DNQX) are structurally distinct from glutamate (see FIG. 10), yet theybind to the iGluR receptor and stimulate or inhibit its action. Thus,any responses which these drugs may effect in plants, are likely to bedue to their specific interaction with a iGluR-type receptor, ratherthan to general effects caused by inhibition of glutamate-utilizingenzymes. This iGluR agonist/antagonist pair can specifically affect theexpression of nitrogen metabolic genes in Arabidopsis.

That plants possess a functional GluR is further supported byApplicants' discovery that specific iGluR against, KA, inhibits plantgrowth and this inhibition can at least partially be reversed byglutamate. A specific iGluR antagonist, DNQX, is able to phenocopyArabidopsis mutants impaired in light and/or hormone signaltransduction. These data combined strongly suggest that plant iGluRs areinvolved in plant signal transduction and may be involved in more thanone signal transduction pathway.

The invention also relates to the use of the plant GluR as a target toselect for new herbicides. The cloned plant GluR described herein can beutilized for the selection of new plant specific herbicides. Glumateanalogs, such as L-methionine-S-sulfoximine (MSO) and phosphonothricin(PPT), are effective herbicides. MSO and PPT may be herbicides actingnot only through target enzymes, but also through GluR.

As shown in the working examples, infra the present invention alsoprovides methods of screening and identifying novel plant growthregulators and pharmaceutical drugs that mimic or antagonize glutamatein regulating plant metabolism, physiology and/or gene expression. Thenovel plant growth regulators may have structural homology to agonistsor antagonists of animal glutamate receptors. Such agonists andantagonists have uses as stimulatory or inhibiting plant growthregulators. Due to their structural homologies with animal glutamatereceptors, plant glutamate receptors proteins and polypeptides can alsobe used in in vitro screening for drugs that act on animal glutamatereceptors.

The methods of identifying these novel plant growth regulators are basedon in vivo screening of chemicals for their abilities to alter plantgrowth, development or gene expression in a manner that can be reversedor enhanced by glutamate or glutamate-antagonists.

In other embodiments, the methods are based on in vitro screening ofchemicals for their abilities to compete or interfere with glutamatebinding of plant or animal GluR.

The present invention also encompasses the use of the cDNA clones of theplant GluR to not only select for new herbicides, but to alsogenetically engineer herbicide resistant plants. Given that glutamateacts as an important signal for growth and development, the inventionalso encompasses modulating the activity of the iGluR and mGluR to altergrowth and development patterns of the plants by techniques such astransgenic plants. Therefore gene constructs encoding the plantglutamate receptor protein and polypeptides can be used in geneticengineering of plants to improve their agronomic or industrialproperties.

The present invention also encompasses the use of plant iGluR mutants toscreen for new drugs affecting the central nervous system (CNS) inhumans. Agonists and antagonists of iGluR receptors in animals are usedas drugs for treating CNS disorders such as Epilepsy, stroke, dementiaand CNS trauma. Arabidopsis mutants super-sensitive to mammalian iGluRagonists or antagonists could potentially be used to screen for newdrugs to treat these types of neurodegenerative diseases in humans.

5.1. The Plant Glutamate Receptor Gene

The present invention encompasses the nucleotide coding sequenceencoding plant GluR proteins and polypeptides. These nucleotidesequences were identified in Arabidopsis and shown to have homology tothe animal glutamate receptor genes, iGluR and mGluR. Additionalnucleotide sequences were identified in Arabidopsis as having a lowdegree of homology to the glutamate binding domain of the glutamatereceptor, which are neither ionotropic nor metabotrobic, are alsodescribed herein. In a specific embodiment described herein, the plantGluR genes were identified by searching the Arabidopsis ExpressedSequence Taq (EST) databand (Newman et al., 1994, Plant Physiol. 106:1241-1255) for cDNAs with identity to the glutamate receptor of animals.The five EST clones that are identified in the present invention werenot previously known to contain a high enough degree of homology toanimal glutamate receptors to be identified as such in the Genebank. Inthe present invention the five EST clones were identified as potentialglutamate receptors due to sequence homology and then furthercharacterized as such as described below.

The cDNA sequence and the deduced amirio acid sequence that encodes aplant glutamate receptor most homologous to the iGluR class of animalglutamate receptors are shown in FIG. 15.

The invention includes nucleic acid One Arabidopsis cDNA clone(EST#107M14T7, pAt-iGR-1) shares high identity to a class of glutamatereceptors called ionotropic (iGluR) receptors which constitutesligand-gated ion channels (Gasic and Hollmann, 1992, Annu. Rev. Physiol.54:507-536; Kanai et al., 1993, TINS 16:365-370; O'Hara et al., 1993,Neuron 11:41-52; Seeburg, 1995, TINS 16:359-365). In animals, binding ofglutamate to membrane bound iGluR stimulates the influx of Ca++resulting in and fast excitatory neurotransmission which willsubsequently cause a wide variety of downstream responses (Gasic andHollmann, 1992, Annu. Rev. Physiol. 54:507-536; Kanai et al., 1993, TINS16:365-370; O'Hara et al., 1993, Neuron 11:41-52; Seeburg, 1995, TINS16:359-365).

A second Arabidopsis cDNA (EST# 97C23T7, pAT-mGR-1) shares identity toanother class of animal glutamate receptors called metabotropicglutamate receptor, mGluR (Gasic and Hollmann, 1992, Annu. Rev. Physiol.54:507-536). In animals, the mGluR class of glutamate receptors iscoupled to a G protein that is linked to inositolphosphate/diacylglycerol formation which results in subsequent releaseof calcium from internal stores (Gasic and Hollmann, 1992, Annu. Rev.Physiol. 54:507-536).

Three additional clones (EST# T20773, EST# ATT50711, EST# ATS2655) wereidentified in Arabidopsis as having a low degree of homology to theglutamate binding domain of the animal glutamate receptor. However,these clones do not correspond to ionotropic nor metabotropic animalGluRs. Therefore these clones may represent a novel class ofglutamate-like receptors in plants. The plant GluR of the presentinvention span the plant cellular membrane, as well as intracellularmembranes including vacuolar membranes, chloroplast membranes andmitochondrial membranes.

The Arabidopsis iGluR cDNA, pAt-iGR-1, shares extensive identity withanimal iGluRs. The plant GluR is unique in that it has homology to bothNMDA-type and KA-type GluR in animals. About 700 nucleotides from the 5′end of the clone pAt-iGR-1 have been sequenced. This clone encodes atruncated peptide which shares an extended region of homology with theionotropic glutamate receptors which covers part of theglutamate-binding site close to transmembrane domain I (TMI) andcontinues through TMII until the end of TMIII (FIG. 5 and FIG. 7A). Thehighest identity to animal iGluR is in the glutamate-binding domain(52%) (FIG. 6). The glutamate-binding region of animal iGluR is a twocleft domain shown by the hatched bars in FIG. 5. The Arabidopsissequence shown in FIG. 7B, corresponds to the first of theseglutamate-binding domains in animal iGluR (FIG. 5). Theglutamate-binding domain shared between animal and plant iGluR has lowbut significant identity to the glutamine-binding domain of an E. colipermease gene (Nakanishi et al., 1990, Neuron 5:569-581) (FIG. 6). Theligand-binding R residue, which is conversed in all ionotropic glutamatereceptors, is also conserved in the putative Arabidopsis iGluR (Kuryatovet al., 1994, Neuron 12:1291-1300).

In non-NMDA type animal iGluRs (kainate-binding iGluR and AMPA iGluR),their mRNAs are subject to RNA editing which modifies their function.For example, in the GluR2 subunit in AMPA type iGluR, RNA editing (Q toR) in TM II (FIG. 1) has been shown to regulate the Ca++ permeability.RNA editing leads to a decrease in Ca++ permeability (Choi, 1988, Neuron1:623-634; Hume et al., 1991, Science 253:1028-1031). In Kainate-Bindingtype iGluR, both the GluR5 and GluR6 subunits also display the Q to Rediting similar to the case of GluR2 of AMPA receptors (Sommer et al.,1991, Cell 67:11-19). GluR6 has two additional positions in TMI that aremodified by RNA editing (Kohler et al., 1993, Neuron 10:491-500). ForGluR6, only when TMI is edited does editing in TMII (Q to R) influenceCa++ permeability (Kohler et al., 1993, Neuron 10:491-500). In contrastto the AMPA receptor channel, GluR6(R) channels edited in TMI show ahigher Ca++ permeability than GluR6(Q) channels (Kohler et al., 1993,Neuron 10:491-500). In the case of NMDA type iGluR, all of the subunitsdo not show RNA editing in TMI and TMII. In fact all subunits contain anN at the site which Q to R editing occurs. NMDA receptors are highlypermeable to Ca++.

Interestingly, the Q/R residue of animal non-NMDA-type ionotropicglutamate receptor which are subject to RNA editing or the correspondingnon-editing. N residue of NMDA-type ionotropic glutamate receptor withinTMII (Seeburg, 1995, TINS 16:359-365), are missing from the predictedpeptide of Arabidopsis pAt-iGR-1. Since these residues are important forthe regulation of permeability of Ca++ ions in animal ionotropicglutamate receptors (Burnashev et al., 1992, Neuron 8:189-198; Kohler etal., 1993, Neuron 10:491-500; Sommer et al., 1991, Cell 67:11-19) theCa++ ion permeability in plant glutamate receptors may be regulated by adifferent mechanism.

The nucleotide sequences encoding Arabidopsis iGluR and mGluR genes canbe used to screen cDNA libraries obtained from Arabidopsis and otherplant species to identify further plant iGluR and mGluR genes. A plantcDNA library may be screened, under conditions of reduced stringency,using a radioactively or nonradioactively labeled fragment of theArabidopsis iGluR and mGluR clones. Alternatively, the Arabidopsis iGlURand mGluR sequences can be used to design degenerate or fully degenerateoligonucleotide probes which can be used as PCR probes or to screenplant cDNA libraries. Alternatively, the probes may be used to screengenomic libraries. As shown by working example, infra, this type ofanalysis has revealed the presence of both iGluR and mGluR genes inother dicots, such as tobacco, a legume, pea, and two monocots, corn andrice. For a review of cloning strategies which may be used, see e.g.,Maniatis 1989 Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, N.Y.

The plant GluR nucleotide sequences of the invention include (a) the DNAsequence shown in FIG. 15; (b) any nucleotide sequence that encodes theamino acid sequence shown in FIG. 15; (c) any nucleotide sequence thathybridizes to the complement of the cDNA sequence shown in FIG. 15 andencodes a functionally equivalent product; (d) any nucleotide sequencethat hybridizes to the complement of of the DNA sequences that encodethe amino acid sequence shown in FIG. 15 and encodes a functionallyequivalent product; (e) any nucelotide sequence encoding a plant proteincontaining the amino acid sequence of the glutamate binding domain shownin FIG. 6; (f) any nucleotide sequence encoding a plant proteincontaining the amino acid sequences shown in FIG. 7A, 7B and/or 7C.Functional equivalents of the plant GluR include naturally occurringplant GluR in other plant species, and mutant GluR whether naturallyoccurring or engineered. The invention also includes degenerate variantsof sequences (a) through (f).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thenucleotide sequences (a) through (f), in the preceding paragraph. Suchhybridization conditions may be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides (“oligos”), highly stringentconditions may refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may encode or act as plant GluR antisensemolecules, useful, for example, in plant GluR gene regulation (forand/or as antisense primers in amplification reactions of plant GluRgene nucleic acid sequences). With respect to plant GluR generegulation, such techniques can be used to regulate, for example, plantgrowth, development or gene expression. Further, such sequences may beused as part of ribozyme and/or triple helix sequences, also useful forplant GluR gene regulation.

In addition to the plant GluR nucleotide sequences described above, fulllength plant GluR cDNA or gene sequences present in the same speciesand/or homologs of the plant GluR gene present in other plant speciescan be identified and readily isolated, without undue experimentation,by molecular biological techniques well known in the art. Theidentification of homologs of plant GluR in related species can beuseful for developing plant model systems for purposes of discoveringplant iGluR agonists or antagonists to modify iGluR in plants to alterthe following processes in either a positive or negative way:germination, growth rate, light-signal transduction and hormone signaltransduction. Alternatively, such cDNA libraries, or genomic DNAlibraries derived from the organism of interest can be screened byhybridization using the nucleotides described herein as hybridization oramplification probes. Furthermore, genes at other genetic loci withinthe genome that encode proteins which have extensive homology to one ormore domains of the plant GluR gene product can also be identified viasimilar techniques. In the case of cDNA libraries, such screeningtechniques can identify clones derived from alternatively splicedtranscripts in the same or different species.

Screening can be by filter hybridization, using duplicate filters. Thelabeled probe can contain at least 15-30 base pairs of the plant GluRnucleotide sequence, as shown in FIG. 15. The hybridization washingconditions used should be of a lower stringency when the cDNA library isderived from an organism different from the type of organism from whichthe labeled sequence was derived.

Low stringency conditions are well known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.;and Ausubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y.

Alternatively, the labeled plant GluR nucleotide probe may be used toscreen a genomic library derived from the organism of interest, again,using appropriately stringent conditions. The identification andcharacterization of plant genomic clones is helpful for designingdiagnostic tests and clinical protocols for regulating plant growthrate, germination, light-signal transduction and hormone signaltransduction. For example, sequences derived from regions adjacent tothe intron/exon boundaries of the plant gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g. splice acceptor and/or donor sites),etc., that can be used in diagnostics.

Further, an plant GluR gene homolog may be isolated from nucleic acid ofthe organism of interest by performing PCR using two degenerateoligonucleotide primer pools designed on the basis of amino acidsequences within the plant GluR gene product disclosed herein. Thetemplate for the reaction may be cDNA obtained by reverse transcriptionof mRNA prepared from, for example, plant cell lines or tissue, known orsuspected to express an plant GluR gene allele.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of a plant GluR gene. ThePCR fragment may then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment may be labeledand used to screen a cDNA library, such as a plant cDNA library.Alternatively, the labeled fragment may be used to isolate genomicclones via the screening of a genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express the plant GluR gene. A reversetranscription reaction may be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” with guanines using a standardterminal transferase reaction, the hybrid may be digested with RNAase H,and second strand synthesis may then be primed with a poly-C primer.Thus, cDNA sequences upstream of the amplified fragment may easily beisolated. For a review of cloning strategies which may be used, seee.g., Sambrook et al., 1989, supra.

The plant GluR gene sequences may additionally be used to isolate mutantplant GluR gene alleles. Such mutant alleles may be isolated from plantspecies either known or proposed to have a genotype which contributes tothe symptoms plant growth rate, germination, light-signal transductionor hormone-signal transduction. Mutant alleles and mutant alleleproducts may then be utilized in the therapeutic and diagnostic systemsdescribed below. Additionally, such plant GluR gene sequences can beused to detect plant GluR gene regulatory (e.g., promoter orpromotor/enhancer) defects which can affect plant growth.

A cDNA of a mutant plant GluR gene may be isolated, for example, byusing PCR, a technique which is well known to those of skill in the art.In this case, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide to mRNA isolated from tissue known or suspectedto be expressed in a plant species putatively carrying the mutant plantGluR allele, and by extending the new strand with reverse transcriptase.The second strand of the cDNA is then synthesized using anoligonucleotide that hybridizes specifically to the 5′ end of the normalgene. Using these two primers, the product is then amplified via PCR,cloned into a suitable vector, and subjected to DNA sequence analysisthrough methods well known to those of skill in the art. By comparingthe DNA sequence of the mutant plant GluR allele to that of the normalplant GluR allele, the mutation(s) responsible for the loss oralteration of function of the mutant plant GluR gene product can beascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom a plant species suspected of or known to carry the mutant plantGluR allele, or a cDNA library can be constructed using RNA from atissue known, or suspected, to express the mutant plant GluR allele. Thenormal plant GluR gene or any suitable fragment thereof may then belabeled and used as a probe to identify the corresponding mutant plantGluR allele in such libraries. Clones containing the mutant plant GluRgene sequences may then be purified and subjected to sequence analysisaccording to methods well known to those of skill in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant plant GluR allele in a plant speciessuspected of or known to carry such a mutant allele. In this manner,gene products made by the putatively mutant tissue may be expressed andscreened using standard antibody screening techniques in conjunctionwith antibodies raised against the normal plant GluR gene product, asdescribed, below, in Section 5.3. (For screening techniques, see, forexample, Harlow, E. and Lane, eds., 1988, “Antibodies: A LaboratoryManual”, Cold Spring Harbor Press, Cold Spring Harbor.) Additionally,screening can be accomplished by screening with labeled GluR fusionproteins. In cases where a plant GluR mutation results in an expressedgene product with altered function (e.g., as a result of a missense or aframeshift mutation), a polyclonal set of antibodies to plant GluR arelikely to cross-react with the mutant plant GluR gene product. Libraryclones detected via their reaction with such labeled antibodies can bepurified and subjected to sequence analysis according to methods wellknown to those of skill in the art.

The invention also encompasses nucleotide sequences that encode mutantplant GluR, peptide fragments of the plant GluR, truncated plant GluR.and plant GluR fusion proteins. These include, but are not limited tonucleotide sequences encoding mutant plant GlUR described in section 5.2infra; polypeptides or peptides corresponding to the signal peptide, twohalves of the putative glutamate-binding domain and the three plus onetransmembrane (TM) domains; truncated plant GluR in which one or two ofthe domains is deleted, e.g., a soluble plant GluR lacking the signalpeptide, the glutamate-binding domain, or the TM domains, or atruncated, nonfunctional plant GluR lacking all or a portion of theglutamate binding domain. Nucleotides encoding fusion proteins mayinclude by are not limited to full length plant GluR, truncated plantGluR or peptide fragments of plant GluR fused to an unrelated protein orpeptide, such as for example, a transmembrane sequence, which anchorsthe plant GluR ECD to the cell membrane or an enzyme, fluorescentprotein, luminescent protein which can be used as a marker.

The invention also encompasses (a) DNA vectors that contain any of theforegoing plant GluR coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingGluR coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences; and (c) geneticallyengineered host cells that contain any of the foregoing plant GluRcoding sequences operatively associated with a regulatory element thatdirects the expression of the coding sequences in the host cell. As usedherein, regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the promoters derivedfrom the genome of plant cells (e.g., heat shock promoters; the promoterfor the small subunit of RUBISCO; the promoter for the chlorophyll a/bbinding protein) or from plant viruses (e.g., the 355 RNA promoter ofCaMV; the coat protein promoter of tobacco mosaic virus (TMV),cytomegalovirus hCMV immediate early gene, the early or late promotersof SV40 adenovirus, the lac system, the trp system, the TAC system, theTRC system, the major operator and promoter regions of phage A, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase, the promoters of acid phosphatase, and the promoters of theyeast α-mating factors.

5.2. Plant GluR Proteins and Polypeptides

Plant GluR protein, polypeptides and peptide fragments, mutated,truncated or deleted forms of the plant GluR and/or plant GluR fusionproteins can be prepared for a variety of uses, including but notlimited to the generation of antibodies, the identification of othercellular gene products involved in the regulation of plant growth, asreagents in assays for screening for compounds that can be used asherbicides, and as pharmaceutical reagents useful in the treatment ofCNS related disorders the plant GluR protein.

FIGS. 15, 6, 7A, 7B, and/or 7C show the amino acid sequence of the plantGluR protein respectively. As shown in FIG. 15 in this form of the plantGluR, the glutamate binding domain 1 spans from amino acid 466 to about537; the TMI spans from amino acid 555 to about 580; the TMII spans fromamino acid 592 to 608; the TMIII spans from about amino acid 614 to 634;the glutamate binding domain 2 spans from about amino acid 714 to 748;and TMIV spans from about amino acid 782 to 809. FIGS. 17 and 18 showthe amino acid sequence alignment with these domains.

The plant GluR sequence begins with a methionine in a DNA sequencecontext consistent with a translation initiation site, followed by atypical hydrophobic signal sequence of peptide secretion.

The plant GluR amino acid sequences of the invention include the aminoacid sequence shown in FIG. 15, FIG. 6 or FIGS. 7A, 7B and/or 7C, or theamino acid sequence encoded by cDNA clone PAT-iGR-1 (EST #107M14T7), orencoded by cDNA clone PAT-mGR-1 (EST #97C23T7). Further, plant GluR ofother plant species are encompassed by the invention. In fact, any plantGluR protein encoded by the plant GluR nucleotide sequences described inSection 5.1, above, are within the scope of the invention.

The invention also encompasses proteins that are functionally equivalentto the plant GluR encoded by the nucleotide sequences described inSection 5.1, as judged by any of a number of criteria, including but notlimited to the ability to bind glutamate, the binding affinity forglutamate, the resulting biological effect of glutamate binding, e.g.,signal transduction, a change in cellular metabolism (e.g., ion flux,tyrosine phosphorylation) or change in phenotype when the plant GluRequivalent is present in an appropriate cell type such as, an increasein plant growth. Such functionally equivalent plant GluR proteinsinclude but are not limited to additions or substitutions of amino acidresidues within the amino acid sequence encoded by the plant GluRnucleotide sequences described, above, in Section 5.1, but which resultin a silent change, thus producing a functionally equivalent geneproduct. Amino acid substitutions may be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity, and/orthe amphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

While random mutations can be made to plant GluR DNA (using randommutagenesis techniques well known to those skilled in the art) and theresulting mutant plant GluRs tested for activity, site-directedmutations of the GluR coding sequence can be engineered (usingsite-directed mutagenesis techniques well known to those skilled in theart) to generate mutant plant GluRs with increased function, e.g.,higher binding affinity for glutamate, and/or greater signallingcapacity; or decreased function, e.g., lower binding affinity forglutamate, and/or decreased signal transduction capacity.

For example, the alignment of animal form of GLuR and the plant GluRhomolog is shown in FIG. 18 in which identical amino acid residues areindicated by vertical lines and conserved residues are indicated bydots. Conservative alterations at the variable positions can beengineered in order to produce a mutant plant GLuR that retainsfunction; e.g., glutamate binding affinity or signal transductioncapability or both. Non-conservative changes can be engineered at thesevariable positions to alter function, e.g., glutamate binding affinityor signal transduction capability, or both. Alternatively, wherealteration of function is desired, deletion or non-conservativealterations of conserved residues in the glutamate binding domain(indicated in FIG. 18) can be engineered. For example, deletion ornon-conservative alterations (substitutions or insertions) of theglutamate binding domains, e.g., amino acid residues 466 to 537 and 714to 748 (FIG. 18) of plant GluR or portions of the glutamate bindingdomain, e.g., amino acid residues 466-537 (FIG. 18) of plant GluR, oramino acid residues 714-748 can be engineered to produce a mutant plantGluR that binds glutamate but is signalling-incompetent.Non-conservative alterations to the residues in the TMs shown in FIG. 18can be engineered to produce mutant plant GluRs with altered bindingaffinity for glutamate.

Other mutations to the plant GluR coding sequence can be made togenerate plant GluRs that are better suited for expression, scale up,etc. in the host cells chosen. For example, cysteine residues can bedeleted or substituted with another amino acid in order to eliminatedisulfide bridges; N-linked glycosylation sites can be altered oreliminated to achieve, for example, expression of a homogeneous productthat is more easily recovered and purified from yeast hosts which areknown to hyperglycosylate N-linked sites. To this end, a variety ofamino acid substitutions at one or both of the first or third amino acidpositions of any one or more of the glycosylation recognition sequenceswhich occur in the extracellular domain (ECD) (N-X-S or N-X-T), and/oran amino acid deletion at the second position of any one or more suchrecognition sequences in the ECD will prevent glycosylation of the plantGluR at the modified tripeptide sequence.

Peptides corresponding to one or more domains of the plant GluR (e.g.,ECD, TM or CD), truncated or deleted plant GluR (e.g., GluR in which theTM and/or CD is deleted) as well as fusion proteins in which the fulllength GluR, a GluR peptide or truncated GluR is fused to an unrelatedprotein are also within the scope of the invention and can be designedon the basis of the GluR nucleotide and GluR amino acid sequencesdisclosed in this Section and in Section 5.1, above. Such fusionproteins include but are not limited to IgFc fusions which stabilize theplant GluR protein or peptide and prolong half-life in vivo; or fusionsto any amino acid sequence that allows the fusion protein to be anchoredto the cell membrane, allowing the ECD to be exhibited on the cellsurface; or fusions to an enzyme, fluorescent protein, or luminescentprotein which provide a marker function.

While the plant GluR polypeptides and peptides can be chemicallysynthesized (e.g., see Creighton, 1983, Proteins: Structures andMolecular Principles, W.H. Freeman & Co., N.Y.), large polypeptidesderived from the GluR and the full length GluR itself may advantageouslybe produced by recombinant DNA technology using techniques well known inthe art for expressing nucleic acid containing plant GluR gene sequencesand/or coding sequences. Such methods can be used to constructexpression vectors containing the plant GluR nucleotide sequencesdescribed in Section 5.1 and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. See, for example, the techniques described inSambrook et al., 1989, supra, and Ausubel et al., 1989, supra.Alternatively, RNA capable of encoding plant GluR nucleotide sequencesmay be chemically synthesized using, for example, synthesizers. See, forexample, the techniques described in “Oligonucleotide Synthesis”, 1984,Gait, M. J. ed., IRL Press, Oxford, which is incorporated by referenceherein in its entirety.

A variety of host-expression vector systems may be utilized to expressthe plant GluR nucleotide sequences of the invention. Where the GluRpeptide or polypeptide is a soluble derivative (e.g., GluR peptidescorresponding to the ECD; truncated or deleted GluR in which the TMand/or CD are deleted) the peptide or polypeptide can be recovered fromthe culture, i.e., from the host cell in cases where the GluR peptide orpolypeptide is not secreted, and from the culture media in cases wherethe GluR peptide or polypeptide is secreted by the cells. However, theexpression systems also encompass engineered host cells that express theplant GluR or functional equivalents in situ, i.e., anchored in the cellmembrane. Purification or enrichment of the plant GluR from suchexpression systems can be accomplished using appropriate detergents andlipid micelles and methods well known to those skilled in the art.However, such engineered host cells themselves may be used in situationswhere it is important not only to retain the structural and functionalcharacteristics of the plant GluR, but to assess biological activity,e.g., in herbicide screening assays.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the plant GluR coding sequenceand appropriate transcriptional/translational control signals. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. See, forexample, the techniques described in Maniatis et al., 1989, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. andAusubel et al., 1989, Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, N.Y.

A variety of host-expression vector systems may be utilized to expressthe plant GluR coding sequence. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the plant GluR coding sequence; yeast transformed withrecombinant yeast expression vectors containing the plant GluR codingsequence; insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing the plant GluR coding sequence;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the plant GluR coding sequence; or animal cellsystems infected with recombinant virus expression vectors (e.g.,adenovirus, vaccinia virus) including cell lines engineered to containmultiple copies of the plant GluR either stably amplified (CHO/dhfr) orunstably amplified in double-minute chromosomes (e.g., murine celllines).

The expression elements of these systems vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedrin promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g., heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter) may be used;when generating cell lines that contain multiple copies of-the plantGluR DNA SV40-, BPV- and EBV-based vectors may be used with anappropriate selectable marker.

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for the plantGluR expressed. For example, when large quantities of plant GluR are tobe produced for the generation of antibodies or to screen peptidelibraries, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include but are not limited to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the plant GluRcoding sequence may be ligated into the vector in frame with the lac Zcoding region so that a hybrid GluR lac Z protein is produced; pINvectors (Inouye & Inouye, 1985, Nucleic acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.pGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the clonedpolypeptide of interest can be released from the GST moiety.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. &Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and Secretionvectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 1987,Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning,Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, HeterologousGene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel,Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology ofthe Yeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring HarborPress, Vols. I and II.

In cases where plant expression vectors are used, the expression of theplant GluR coding sequence may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S RNA and 19S RNApromoters of CaMV (Brisson et al., 1984, Nature 310:511-514), or thecoat protein promoter of TMV. (Takamatsu et al., 1987, EMBO J.6:307-311) may be used; alternatively, plant promoters such as the smallsubunit of RUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglieet al., 1984, Science 224:838-843); or heat shock promoters, e.g.,soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol.6:559-565) may be used. These constructs can be introduced into plantcells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNAtransformation, microinjection, electroporation, etc. For reviews ofsuch techniques see, for example, Weissbach & Weissbach, 1988, Methodsfor Plant Molecular Biology, Academic Press, NY, Section VIII, pp.421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed.,Blackie, London, Ch. 7-9.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe plant GluR may be engineered. Rather than using expression vectorswhich contain viral origins of replication, host cells can betransformed with the plant GluR DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the plantGluR on the cell surface, and which respond to glutamate mediated signaltransduction. Such engineered cell lines are particularly useful inscreening glutamate analogs.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl.. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin genes (Santerre, et al., 1984, Gene 30:147). Recently,additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman &Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); and ODC (ornithinedecarboxylase) which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987,In: Current Communications in Molecular Biology, Cold Spring HarborLaboratory ed.). The invention also encompasses (a) DNA vectors thatcontain any of the foregoing coding sequences and/or their complements(i.e., antisense); (b) DNA expression vectors that contain any of theforegoing coding sequences operatively associated with a regulatoryelement that directs the expression of the coding sequences; and (c)genetically engineered host cells and/or plants that contain any of theforegoing coding sequences operatively associated with a regulatoryelement that directs the expression of the coding sequences in the hostcell. As used herein, regulatory elements include but are not limited toinducible and non-inducible promoters, enhancers, operators and otherelements known to those skilled in the art that drive and regulateexpression.

In addition to the gene sequences described above, homologues of suchsequences, as may, for example, be present in other plant species may beidentified and may be readily isolated, without undue experimentation,by molecular biological techniques well known in the art. Further, theremay exist genes at other genetic loci within the genome that encodeproteins which have extensive homology to one or more domains of suchgene products. These genes may also be identified via similartechniques.

For example, the isolated GluR gene sequence may be labeled and used toscreen a cDNA library constructed from mRNA obtained from the organismof interest. The hybridization conditions used should be of a lowerstringency when the cDNA library is derived from an organism differentfrom the type of organism from which the labeled sequence was derived.Alternatively, the labeled fragment may be used to screen a genomiclibrary derived from the organism of interest, again, usingappropriately stringent conditions. Low stringency conditions are wellknown to those of skill in the art, and will vary predictably dependingon the specific organisms from which the library and the labeledsequences are derived. For guidance regarding such conditions see, forexample, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,Cold springs Harbor Press, N.Y.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y.

Further, previously unknown GluR gene-type sequences may be isolated byperforming PCR using two degenerate oligonucleotide primer poolsdesigned on the basis of amino acid sequences within the gene ofinterest. The template for the reaction may be cDNA obtained by reversetranscription of mRNA prepared from cell lines or tissue known orsuspected to express an GluR gene allele.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of an GluR gene-like nucleicacid sequence. The PCR fragment may then be used to isolate a fulllength cDNA clone by a variety of methods. For example, the amplifiedfragment may be labeled and used to screen a bacteriophage cDNA library.Alternatively, the labeled fragment may be used to screen a genomiclibrary.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source. A reversetranscription reaction may be performed on the RNA using anoligonucleotide primer specific for the most 5′end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” with guanines using a standardterminal transferase reaction, the hybrid may be digested with RNAase H,and second strand synthesis may then be primed with a poly-C primer.Thus, cDNA sequences upstream of the amplified fragment may easily beisolated. For a review of cloning strategies which may be used, seee.g., Sambrook et al., 1989, supra.

In cases where the GluR gene identified is the normal, or wild typegene, this gene may be used to isolate mutant alleles of the gene.Mutant alleles may be isolated from plants either known or proposed tohave a genotype which contributes abnormal growth characteristics.Mutant alleles and mutant allele products may then be utilized in thedevelopment of in vitro assays, plant assay systems, and transgenicplants as described below.

A cDNA of the mutant gene may be isolated, for example, by using PCR, atechnique which is well known to those of skill in the art. In thiscase, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide to mRNA isolated from tissue known or suspectedto be expressed in an individual putatively carrying the mutant allele,and by extending the new strand with reverse transcriptase. The secondstrand of the cDNA is then synthesized using an oligonucleotide thathybridizes specifically to the 5′ end of the normal gene. Using thesetwo primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant gene to that of the normal gene, the mutation(s)responsible for the loss or alteration of function of the mutant geneproduct can be ascertained.

Alternatively, a genomic or cDNA library can be constructed and screenedusing DNA or RNA, respectively, from plant cells, tissues or wholeplants suspected of expressing the gene of interest in an individualsuspected of or known to carry the mutant allele. The normal gene or anysuitable fragment thereof may then be labeled and used as a probed toidentify the corresponding mutant allele in the library. The clonecontaining this gene may then be purified and subjected to sequenceanalysis according to methods well known to those of skill in the art.

Additionally, an expression library can be constructed utilizing DNAisolated from or cDNA synthesized from plant cells, tissues or wholeplants suspected of expressing the gene of interest in an individualsuspected of or known to carry the mutant allele. In this manner, geneproducts made by the putatively mutant tissue may be expressed andscreened using standard antibody screening techniques in conjunctionwith antibodies raised against the normal gene product, as described,below, in Section 5.3. (For screening techniques, see, for example,Harlow, E. and Lane, eds., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Press, Cold Spring Harbor.) In cases where the mutationresults in an expressed gene product with altered function (e.g., as aresult of a missense mutation), a polyclonal set of antibodies arelikely to cross-react with the mutant gene product. Library clonesdetected via their reaction with such labeled antibodies can be purifiedand subjected to sequence analysis according to methods well known tothose of skill in the art.

5.2.1. Identification of Transfectants or Transformants Expressing thePlant GluR Gene Product

The host cells which contain the plant GluR coding sequence and whichexpress the biologically active gene product may be identified by atleast four general approaches: (a) DNA-DNA or DNA-RNA hybridization; (b)the presence or absence of “marker” gene functions; (c) assessing thelevel of transcription as measured by the expression of plant GluR mRNAtranscripts in the host cell; and (d) detection of the gene product asmeasured by immunoassay or by its biological activity.

In the first approach, the presence of the plant GluR coding sequenceinserted in the expression vector can be detected by DNA-DNA or DNA-RNAhybridization using probes comprising nucleotide sequences that arehomologous to the plant GluR coding sequence or portions or derivativesthereof.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine activity, resistance toantibiotics, resistance to methotrexate, transformation phenotype,occlusion body formation in baculovirus, etc.). For example, if theplant GluR coding sequence is within a marker gene sequence of thevector, recombinants containing the plant GluR coding sequence can beidentified by the absence of the marker gene function. Alternatively, amarker gene can be placed in tandem with the plant GluR sequence underthe control of the same or different promoter used to control theexpression of the plant GluR coding sequence. Expression of the markerin response to induction or selection indicates expression of the plantGluR coding sequence.

In the third approach, transcriptional activity for the plant GluRcoding region can be assessed by hybridization assays. For example, RNAcan be isolated and analyzed by Northern blot using a probe homologousto the plant GluR coding sequence or particular portions thereofsubstantially as shown in FIGS. 6 and 7. Alternatively, total nucleicacids of the host cell may be extracted and assayed for hybridization tosuch probes.

In the fourth approach, the expression of the plant GluR protein productcan be assessed immunologically, for example by Western blots,immunoassays such as radioimmuno-precipitation, enzyme-linkedimmunoassays and the like. The ultimate test of the success of theexpression system, however, involves the detection of the biologicallyactive plant GluR gene product. Where the host cell secretes the geneproduct, the cell free media obtained from the cultured transfectanthost cell may be assayed for plant GluR activity. Where the gene productis not secreted, cell lysates may be assayed for such activity. Ineither case, a number of assays can be used to detect plant GluRactivity including but not limited to the following: cycloxygenaseactivity may be determined in the culture medium by the addition ofexogenous arachidonic acid substrate (30 μM for 15 min. at 37° C.)followed by conversion of the prostayalandin E₂₀ product to a methyloximate form. This bicyclic derivative may then be quantitated byradioimmunoassay (kit from Amersham Corp).

Desired plants and plant cells may be obtained by engineering the geneconstructs described herein into a variety of plant cell types,including but not limited to, protoplasts, tissue culture cells, tissueand organ explants, pollen, embryos as well as whole plants. In anembodiment of the present invention, the engineered plant material isselected or screened for transformants (i.e., those that haveincorporated or integrated the introduced gene construct(s)) followingthe approaches and methods described below. An isolated transformant maythen be regenerated into a plant. Alternatively, the engineered plantmaterial may be regenerated into a plant or plantlet before subjectingthe derived plant or plantlet to selection or screening for the markergene traits. Procedures for regenerating plants from plant cells,tissues or organs, either before or after selecting or screening formarker gene(s), are well known to those skilled in the art.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection may be performed by growing the engineered plantmaterial on media containing inhibitory amounts of the antibiotic orherbicide to which the transforming marker gene construct confersresistance. Further, transformed plants and plant cells may also beidentified by screening for the activities of any visible marker genes(e.g., the β-glucuronidase, luciferase, B or C1 genes) that may bepresent on the recombinant nucleic acid constructs of the presentinvention. Such selection and screening methodologies are well known tothose skilled in the art.

Physical and biochemical methods also may be used to identify a plant orplant cell transformant containing the gene constructs of the presentinvention. These methods include but are not limited to: 1) Southernanalysis or PCR amplification for detecting and determining thestructure of the recombinant DNA insert; 2) Northern blot, S-1 RNaseprotection, primer-extension or reverse transcriptase-PCR amplificationfor detecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) protein gelelectrophoresis, western blot techniques, immunoprecipitation, orenzyme-linked immunoassays, where the gene construct products areproteins; 5) biochemical measurements of compounds produced as aconsequence of the expression of the introduced gene constructs.Additional techniques, such as in situ hybridization, enzyme staining,and immunostaining, also may be used to detect the presence orexpression of the recombinant construct in specific plant organs andtissues. The methods for doing all these assays are well known to thoseskilled in the arts.

5.2.2. Purification of the Plant GluR Gene Product

Once a cell that produces high levels of biologically active plant GluRis identified, the cell may be clonally expanded and used to producelarge quantities of the receptor. The receptor may be purified usingtechniques well-known in the art including, but not limited to,immunoaffinity purification, chromatographic methods including highperformance liquid chromatography and the like. Where the gene productis secreted by the cultured cells, plant GluR polypeptides or peptidesmay be readily recovered from the culture medium.

Where the plant GluR coding sequence has been engineered to encode acleavable fusion protein, the purification of lant GluR may be readilyaccomplished using affinity purification techniques. For example, anantibody specific for the heterologous peptide or protein can be used tocapture the durable fusion protein; for example, on a solid surface, acolumn etc. The plan GluR moiety can be released by treatment with theappropriate enzyme that cleaves the linkage site.

The ease of cDNA construction using the polymerase chain reaction,transfection and purification of the expressed protein permits theisolation of small, but sufficient amount of plant GluR forcharacterization of the receptor's physical and kinetic properties.Using site-directed mutagenesis or naturally occurring mutant sequences,this system provides a reasonable approach to determine the effects ofthe altered primary structure on the function of the protein. Fusionconstructs having the domain of plant GluR preceding the amino terminusof the cleavable protein versus constructs having the oppositearrangement, may also be engineered to evaluate which fusion constructwill interfere the least, if at all, with the protein's biologicfunction and the ability to be purified.

Using this aspect of the invention, any cleavage site or enzyme cleavagesubstrate may be engineered between the plant GluR sequence and a secondpeptide or protein that has a binding partner which could be used forpurification, e.g, any antigen for which an immunoaffinity column can beprepared.

5.3. Antibodies to GluR Proteins

Antibodies that define the GluR gene product are within the scope ofthis invention, and include antibodies capable of specificallyrecognizing one or more GluR gene product epitopes. Such antibodies mayinclude, but are not limited to, polyclonal antibodies, monoclonalantibodies (mAbs), humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, fragments produced by aFab expression library, anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. Such antibodies may beused, for example, in the detection of an GluR gene product in abiological sample, including, but not limited to, blood plasma andserum. Alternatively, the antibodies may be used as a method for theinhibition of abnormal GluR gene product activity.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to produce single chainantibodies against GluR gene products. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

5.4. Transgenic Plants that Express Mutant GluR

Glutamate in plants may also act as an important signal for growth anddevelopment. It is therefore possible to alter the growth anddevelopment patterns of the plants by modulating the iGluR or mGluR orglutamate-like receptor activities by engineering transgenic plantsexpress either wild type or mutant forms of plant GluR as discussed inSections 5.1 and 5.2. Glutamate has potential activity in regulatingcircadium rythym, therefore it is possible to engineer transgenic plantsexpressing mutated GluR to alter the plant's response to light and thebiological clock. Thus, for example it would be possible to engineerplants which flower early or later, etc. The overexpression of AS and GSgenes results in plants with excellent growth traits, that is, they growfaster and larger. Therefore, it is possible by altering the GluR tostimulate over expression of the AS gene, to genetically engineer plantswith similar growth traits. In addition, by altering the GluR ingenetically engineered cells it may also be possible to synchronizecells in culture, as well as synchronizing plants and seed germination.

According to the present invention, a desirable plant or plant cell maybe obtained by transforming a plant cell with the nucleic acid.constructs described in Section 5.1. In some instances, it may bedesirable to engineer a plant or plant cell with several different geneconstructs. Such engineering may be accomplished by transforming a plantor plant cell with all of the desired gene constructs simultaneously.Alternatively, the engineering may be carried out sequentially. That is,transforming with one gene construct, obtaining the desired transformantafter selection and screening, transforming the transformant with asecond gene construct, and so on.

In an embodiment of the present invention, Agrobacterium can be employedto introduce the gene constructs into plants. Such transformationspreferably use binary Agrobacterium T-DNA vectors (Bevan, 1984, Nuc.Acid Res. 12:8711-8721), and the co-cultivation procedure (Horsch etal., 1985, Science 227:1229-1231). Generally, the Agrobacteriumtransformation system is used to engineer dicotyledonous plants (Bevanet al., 1982, Ann. Rev. Genet 16:357-384; Rogers et al., 1986, MethodsEnzymol. 118:627-641). The Agrobacterium transformation system may alsobe used to transform as well as transfer DNA to monocotyledonous plantsand plant cells. (see Hernalsteen et al., 1984, EMBO J 3:3039-3041 ;Hooykass-Van Slogteren et al., 1984, Nature 311:763-764; Grimsley etal., 1987, Nature 325:1677-179; Boulton et al., 1989, Plant Mol. Biol.12:31-40.; Gould et al., 1991, Plant Physiol. 95:426-434).

In other embodiments, various alternative methods for introducingrecombinant nucleic acid constructs into plants and plant cells may alsobe utilized. These other methods are particularly useful where thetarget is a monocotyledonous plant or plant cell. Alternative genetransfer and transformation methods include, but are not limited to,protoplast transformation through calcium-, polyethylene glycol (PEG)-or electroporation-mediated uptake of naked DNA (see Paszkowski et al.,1984, EMBO J 3:2717-2722, Potrykus et al. 1985, Molec. Gen. Genet.199:169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82:5824-5828;Shimamoto, 1989, Nature 338:274-276) and electroporation of planttissues (D'Halluin et al., 1992, Plant Cell 4:1495-1505). Additionalmethods for plant cell transformation include microinjection, siliconcarbide mediated DNA uptake (Kaeppler et al., 1990, Plant Cell Reporter9:415-418), and microprojectile bombardment (see Klein et al., 1988,Proc. Nat. Acad. Sci. USA 85:4305-4309; Gordon-Kamm et al., 1990, PlantCell 2:603-618).

According to the present invention, a wide variety of plants and plantcell systems may be engineered for the desired physiological andagronomic characteristics described herein using the nucleic acidconstructs of the instant invention and the various transformationmethods mentioned above. In preferred embodiments, target plants andplant cells for engineering include, but are not limited to, those ofmaize, wheat, rice, soybean, tomato, tobacco, carrots, peanut, potato,sugar beets, sunflower, yam, Arabidopsis, rape seed, and petunia.

The present invention also encompasses the use of the cDNA clones of theplant GluR to not only select for new herbicides, but to alsogenetically engineer herbicide resistant plants. Gene constructsencoding the plant glutamate receptor protein and polypeptides can beused in genetic engineering of plants to alter the plant's growthrequirements or conditions. The invention also encompasses geneticallyengineered plants that express mutagenized forms of the plant GluR sothat the plants are able to utilize alternative nitrogen sources, whichmay prove to be beneficial, for example in improving plant growth innitrogen limited soil. Given the important role that glutamate signalingplays in timing of cell division or flowering, transgenic plants may beengineered to produce more flowers or fruit in response to a specificnitrogen source. Therefore the present invention has many utilitiesin.genetic engineering of plants to improve their agronomic orindustrial properties.

The invention further encompasses transgenic plants expressing chimericGluR. By exchanging portions of the glutamate binding domain withsegments of receptors sensitive to other compounds it is possible toengineer a receptor with specific resistance or growth response to thatspecific compound. Animal GluR subunits with specificity to particularagonists have been identified. (Bach et al., 1994, Neuron 13:1343-1357). Through mutagenesis of the plant GluR it is possible toidentify similar segments in plants and to genetically engineer GluRresponsive to one specific agonist or analog of glutamate. Transgenicplants expressing such receptors would have improved agronomic andindustrial properties.

5.5. Screening Assays for Herbicides

The present invention also encompasses screening assays to identifyagonists and antagonists of glutamate induced signaling. This inventionprovides a novel, rapid and cost effective means of screening oridentifying novel plant growth regulators and pharmaceutical drugs. Themethods are based in some cases on in vivo screening of drugs for theirabilities to alter plant growth, development or gene expression in amanner that can be reversed or enhanced by glutamate orglutamate-antagonists. In other embodiments, the methods are based on invitro screening of drugs for their abilities to compete or interferewith glutamate binding of plant or animal glutamate receptors. Thesemethods may be used to identify novel stimulatory or inhibitory plantgrowth regulatory compounds.

5.5.1. In Vitro Assays

The present invention also encompasses the use of in vitro screens toidentify drugs for their ability to interefere with glutamate binding ofplant or animal glutamate receptors. A potential method for in vitroscreening involves linking the isolated plant glutamate receptor to asolid matrix, such as a sepharose. A solution containing glutamate alongwith the specific drug in varying concentrations. After several washes,the amount of glutamate bound to the receptor can be measured byapplying a radioactively or fluorescently tagged antibody directed tothe glutamate binding domain. The effectiveness of the inhibitors wouldbe measured by the amount of antibody in the flow through. This would bea very rapid and cost effective assay to initially screen for potentialinhibitors of glutamate bindinding to its receptor.

5.5.2. Cell Culture Assays

The present invention encompasses the use of cell lines, that expressthe plant GluR, in in vitro assays to screen drugs for their effects onplant growth mediated by the GluR. Preferably, continuous cell linesstably expressing the GluR gene product or mutants thereof as describedabove and preferably which respond to the signal generated by glutamatebinding are utilized in assays to ideatify novel drugs that either mimicthe effects of glutamate or act as glutamate antagonists. Cell linesthat may be used in the cell culture assay include any cells, includinganimal, human or plant cells, that are engineered (as described inSection 5) to express the plant GluR. Glutamate receptor gene productsencoding plant iGluR, mGluR, glutamate-like receptors, or any otherglutamate receptor expressed in plants, as described in Section 5.1. maybe expressed in cells for use in cell culture assay system.

In this assay, cells expressing the GluR in culture mdium will betreated with potential agonists and antagonists. These drugs will beadded in serial dilution to the culture medium. The effects of thesedrugs will be measured as changes in cell metabolism or growth, changesin gene expression, changes in downstream signaling events and changesin membrane potential. The effects of the drugs should be reversed byglutamate supplemention, which idnicates that the inhibitory effects arespecific to the glutamate-related process.

The cell culture assay will include cells expressing glutamateresponsive gene promoters linked to reporter genes. AS and GS genes areinduced in response to glutamate binding its receptor. Therefore, drugscan be tested for their ability to activate the GluR by induction of ASand GS genes. Induction of gene expression will be measured by linkingthe AS and GS promoters to reporter genes, such as chloramphenicoltransferese, which are commonly used in the art. Induction of the GluRwill be rapdily measured by assaying for the reporter genes activity.

The cell culture assay will also include assaying for GluR activators orinhibitors by measuring an electrophysiological response. Cells, i.e.Hela cells, expressing the plant GluR can be used to assay forinhibitors or activators of the GluR by measuring changes in membranepotential. Ionotropic GluR are known to be ion gated thereforeactivation or inhibition of the GluR may be measured as a change inmembrane potential by methods well known to those skilled in the art.

The in vitro cell assay will provide a very rapid method to screenpotential agonists and antagonists of the GluR. The use of cell linesexpressing mutants of the GluR will provide more information regardingthe action of these compounds. Primary cell cultures, protoplast and/orcell lines stably expressing the GluR which also express mutations inthe signal transduction pathways will be a very useful tool indetermining the downstream signaling events. For example, cell linesstably expressing the GluR may also express a mutant of the plant rashomolog or the plant mitogen-activated kinase (MAPK) homolog in order todetermine whether ras or MAPK is required by glutamate mediated effectson cell metabolism and gene expression. Changes in MAPK activity orphosphorylation may also be utilized as an indicator of GluR activation.

This in vitro assay system is useful for screening and identifyingpotential inhibitors or activators of signaling cascades activated byplants. Cell lines expressing mutagenized GluR will provide a means ofidentifying inhibitors of GluR activated signaling events which may bepotential herbicides. The effects or inhibitors or activators may beobserved by measuring changes in gene expression and phosphorylationevents by methods well known by those skilled in the art.

In another embodiment, the methods are based on in vitro screening ofdrugs for their abilities to compete or interfere with glutamate bindingof animal glutamate receptors. Therefore the invention also encompassesplant cells expressing both wild-type and mutant forms of the animalGluR. In order to perform cell culture assays for drugs which wouldstimulate or inhibit animal glutamate receptors.

5.5.3. Plant Growth Assay

In order to screen for potential plant growth regulators, assaysmeasuring changes in plant growth in response to potential GluR agonistsand antagonists in encompassed by the present invention. Assay measuringchanges in plant growth include changes in root, stem, or leaf growth.The use of transgenic plants as described in Section 5.4. is alsoincluded.

The following assay may be utilized in order to screen drugs for theireffects on plant growth mediated by the GluR. Arabidopsis seedlingsexpressing or overexpressing the GluR are treated with the potentialagonist or antagonist. In this plant growth assay, Arabidopsis seeds areplated on tissue culture plates in MS Medium (Murashige and Skoog SaltMixture-plant basic medium available from Gibco (BRL)). A dose-responsecurve is determined using various concentrations of the potentialagonist or antagonist added to the medium. The plants are grownvertically in a growth chamber at 22° C. with a 16 hour light/8 hourdark cycle for two weeks. The effects of each herbicide on plant growthis assessed by measuring root length on vertical tissue culture plates.The effectiveness of the drug is measured by a increase or reduction inroot growth. The effect of the drug should be reversed by glutamatesupplementation, which indicates that the inhibitory effects arespecific to a glutamate-related process.

This assay also has utility in the selection of new pharmacologicalagonists and antagonist for use as human drugs. The pathophysiologicalinvolvement of GluR receptors in animals has been reviewed (Gasic etal., 1992, Annu. Rev. Physiol. 54: 507-536). As summarized by Gasic andHollmann (1992), glutamate receptors in animals are involved in CNSdisorders such as Huntington's disease, Parkinson's disease, andAlzheimer's disease. GluR is also involved in the initiation andpropagation of seizures and in massive neuronal cell death duringperiods of ischemia and hypoglycemia. It has also been reported thatNMDA receptor antagonists may confer protection to some neurotoxicity inan experimental model of Parkinson's disease (Graham et al. 1990, LifeSci 47:PL91-PL97). Moreover, both non-competitive NMDA receptorantagonist (dizocilpine) and competitive NMDA receptor antagonist(DlL-(E)-2-amino-4-methyl-5-phosphono-3-pentonoic acid) may act asantidepressant (Papp et al., 1994, Eur. J. Pharmacol. 263:1-7). IsolatedArabidopsis mutants which are supersensitive to GluR agonists orantagonists will be utilized to screen for new pharmaceutical drugs suchas new antidepressants. The bioassay for new GluR acting drugs would besensitive, rapid, and cost-effective to operate.

This assay will also have utility to screen and identify potentialinhibitors or activators of signaling cascades activated by the plantGluR. The activation of the GluR leads to activation of downstreamsignaling events which are required for the observed changes in plantmetabolism and development. Therefore the mutagenized Arabidopsisseedlings will have utility in assaying potential inhibitors oractivators of the GluR activated signaling events. The effects of theinhibitors or activators will be observed by measuring changes in rootlength on vertical tissue plates.

5.6. Herbicides that Block the GluR Signal

A further aspect of the invention relates to novel plant growthregulators that mimic or antagonize glutamate in regulating plantmetabolism, physiology and/or gene expression. The novel plant growthregulators may have structural homology to agonists or antagonists ofanimal glutamate receptors. Plant growth regulators that mimic orantagonize the GluR may also be useful in synchronizing plantdevelopment and seed germination, as well as synchronizing cells inculture. The novel plant growth regulators may also have activities asagonists or antagonists of animal glutamate receptors.

Glutamate analogs should be effective herbicide targets, given that theyare all acting not only through some target enzymes, but through plantamino acids receptors which signal downstream responses. The short-termand long-term herbicidal effects on the downstream reactions of thesereceptors would be more profound than the inhibition of a singlebiochemical reaction. The suggestion that some herbicides can affect awhole series of downstream responses have been implicated in Balke'sreview (Balke, 1985, In: Weed Physiology. CRC Press, Inc., p113-139).Several herbicides are suggested to be able to affect the hormonal andenvironmental regulation of membrane functions. For example, both theauxin and auxin antagonist herbicides affect membrane functions that areassociated with the action of IAA in plant cells. Several herbicides canalso affect the phytochrome-regulated Ca++ transport and blue lightinduced absorbance change. However, the detailed mechanisms of theseherbicidal effects are yet to be fully elucidated and the correspondingreceptors of the signals are poorly understood.

The present invention also includes plant growth regulators, includingherbicides that prevent glutamate from binding to the glutamatereceptor. Potential herbicides include, but are not limited to, peptidescorresponding to extracellular domain of the glutamate receptor. Thesepeptides would bind to glutamate and prevent binding to the receptor.

The present invention also includes herbicides that would not only actby binding to the GluR, but rather act intracellularly to inhibitdownstream signaling pathways. Activation of the plant GluR results inthe activation of signaling cascades. These signaling cascades andinhibitors thereof are well characterized in animals. Therefore giventhe strong identity between the animal and plant glutamate receptors, itis likely that similar signaling pathways are activated downstream andtherefore inhibitors of these pathways in animal cells would haveutility in plants. Therefore potential herbicides include, but are notlimited to, inhibitors of plant homologues of ras, raf, protein tyrosinekinases, protein tyrosine phosphatases. Potential herbicides alsoinclude reagents which would inhibit intracellular activation of theglutamate receptor therefore blocking activation of downstream signalingcascades.

Herbicides which act via the GluR signaling pathway but are too large tocross the blood-brain barrier are especially preferred since they wouldbe relatively non-toxic to humans and animals. For example, glutamateanalogs such as PPT in theory should also be toxic to animals, asanimals possess GS and glutamate receptors. However, PPT is not toxic toanimals, as it does not pass the blood-brain barrier and effectivelyexcreted in kidneys. This indicates that there may be plenty of room forconstructing a herbicide which only affects plants but not animals. Inaddition, since the plant iGluR described herein appears to be “novel”in structure (combines kainate and NMDA domains), it is possiblydistinct from those animal iGluRs and may be used to developplant-specific herbicides.

In examples described below, glutamate is shown to induce the expressionof a gene for asparagine synthetase (ASN1) in Arabidopsis thaliana.Thus, glutamate, an amino acid thought to be involved in nitrogentransport, can also act as a signaling molecule in plants. ArabidopsiscDNAs with high identity to a ionotropic animal glutamate receptor(iGluR) as well as one with high identity to animal metabotropicglutamate receptor (mGluR) are identified herein. The data describedbelow shows that kainate, an iGluR agonist, can mimic theglutamate-stimulated induction of ASN1 mRNA. Conversely, an iGluRantagonist (DNQX) partially blocks the induction of ASN1 mRNA byglutamate. These data indicate that Arabidopsis possesses a functionaliGluR.

6. EXAMPLE Identification and Characterization of Arabidopsis Homologsof the Animal Glutamate Receptor Gene

Two Arabidopsis cDNA clones contained in the Arabidopsis ExpressedSequence Tag (EST) databank (Newman et al., 1994, Plant Physiol.106:1241-1255). Two cDNA clones each with identity to a distinct type ofanimal glutamate receptor. One Arabidopsis cDNA clone (EST#107M14T7,pAt-iGR-1) shares high identity to a class of glutamate receptors calledionotropic (iGluR) receptors which constitutes ligand-gated ion channels(Gasic, G. P. and Hollmann, 1992, Annu. Rev. Physiol. 54:507-536; Kanaiet al., 1993, TINS 16:36-370; O'Hara et al., 1993, Neuron 11:41-52;Seeburg, Ph.D., 1995, TINS 16:359-365. A second Arabidopsis cDNA (EST#97C23T7, pAT-mGR-1) shares identity to another class of animal glutamatereceptors called metabotropic glutamate receptor, mGluR (Gasic, G. P.and Hollmann, 1992, Annu. Rev. Physiol. 54:507-536).

6.1. The iGluR cDNA Clone

Sequence analysis of each clone indicates that the Arabidopsis iGluRcDNA, pAt-iGR-1, shares extensive identity with animal iGluRs. About 700nucleotides from the 5′ end of the clone pAt-iGR-1 have been sequenced.This clone encodes a truncated peptide which shares an extended regionof homology with the ionotropic glutamate receptor which cover part ofthe glutamate-binding site close to transmembrane domain I (TMI) andcontinues through TMII until the end of TMIII (FIG. 5 and FIG. 7A). Thehighest identity to animal iGluR is in the glutamate-binding domain(52%) (FIG. 6). The glutamate-binding region of animal iGluR is a twocleft domain shown by the hatched bars in FIG. 5. The Arabidopsissequence shown in FIG. 6, corresponds to the first of theseglutamate-binding domains in animal iGluR (FIG. 5). Theglutamate-binding domain shared between animal and plant iGluR has lowbut significant identity to the glutamine-binding domain of an E. colipermease gene (Nakanishi et al., 1990, Neuron 5:569-581) (FIG. 6). Theligand-binding R residue, which is conserved in all ionotropic glutamatereceptors, is also conserved in the Arabidopsis iGluR (Kuryatov et al.,1994, Neuron 12:1291-1300).

The mRNAs of non-NMDA type iGluRs (kainate-binding iGluR and AMPA iGluR)are subject to RNA editing which modifies their function. For example,in the GluR2 subunit in AMPA type iGluR, RNA editing (Q to R) in TM II(FIG. 1) has been shown to regulate the Ca++ permeability. RNA editingleads to a decrease in Ca++ permeability (Choi, D. W., 1988, Neuron1:623-634; Hume et al., 1991, Science 253:1028-1031). In Kainate-bindingtype iGluR, both the GluR5 and GluR6 subunits display Q to R editingsimilar to that of the GluR2 subunit of AMPA receptors (Sommer et al.,1991, Cell 67:11-19). GluR6 has two additional positions in TMI that aremodified by RNA editing (Kohler et al., 1993, Neuron 10:491-500). ForGluR6, only when TMI is edited does editing in TMII (Q to R) influenceCa++ permeability (Kohler et al., 1993, Neuron 10:491-500). In contrastto the AMPA receptor channel, GluR6(R) channels edited in TMI show ahigher Ca++ permeability than GluR6(Q) channels (Kohler et al., 1993,Neuron 10:491-500). In the case of NMDA type iGluR, all of the subunitsdo not show RNA editing in TMI and TMII. In fact all subunits contain anN at the site at which Q to R editing occurs. NMDA receptors are highlypermeable to Ca++.

Interestingly, the Q/R residue of non-NMDA-type ionotropic glutamatereceptor which are subject to RNA editing or the correspondingnon-editing N residue of NMDA-type ionotropic glutamate receptor withinTMII (Seeburg, P. H., 1995, TINS 16:359-365), are missing from thepredicted peptide of Arabidopsis pAt-iGR-1. Since these residues areimportant for the regulation of permeability of Ca++ ions in animalionotropic glutamate receptors (Burnashev et al., 1992, Neuron8:189-198; Kohler et al., 1993, Neuron 10:491-500; Sommer et al., 1991,Cell 67:11-19) the Ca++ ion permeability in plant glutamate receptorsmay be regulated by a different mechanism.

Sequence searches of GeneBank data using FASTA program show that thepeptide encoded by Arabidopsis pAt-iGR-1 has a strong homology to all ofthe kainate-binding, NMDA, and AMPA types of animal iGluRs, although itseems to have a slightly higher overall homology to kainate-binding typeof iGluRs. FIG. 7A shows the extensive homology between the peptideencoded by the first 700 nucleotides of Arabidopsis pAt-iGR-1 to a NMDAand a kainate-binding iGluR (FIG. 7A). In contrast to animals which havedistinct kainate and NMDA receptors, plants may possess a “novel” typeof iGluR that combines domains of kainate and NMDA receptors.

Northern blot analysis of leaf, root and flower tissues show that theiGluR mRNA is expressed at detectable levels (FIG. 9). In theseexperiments twenty μg of total RNA isolated from each of the leaf, rootand flower tissues were electrophoresed on a 1% formaldehyde agarosegel. The Northern blot analyses were performed with high-stringencyconditions at a temperature of 42° C. in 50% (v/v) formamidehybridization solution. Washing and chemiluminescent detection wereperformed according to the Boehringer-Mannheim Genius System User'sGuide. This Northern shows iGluR mRNA is detected in 20 μg of total RNA.This Northern blot analysis also shows that Arabidopsis iGluR in RNA(2.7 Kb) is of comparable size to animal iGluR mRNA (3.0 Kb) (FIG. 8).The Arabidopsis iGluR mRNA is expressed predominantly in leaves and alsoat lower levels in roots and flowers of Arabidopsis.

6.2. The mGluR cDNA Clone

The Arabidopsis clone pAT-mGR-1 (EST #97C23T7) shares a high degree ofhomology to an animal mGluR as shown in FIG. 7B. The homologous regionbetween Arabidopsis mGluR and animal mGluR includes part of thecysteine-rich region of animal mGluR and spans part of the 7transmembrane domains (O'Hara et al., 1993, Neuron 11:41-52). In theArabidopsis pAt-mGR-1 clone, there are several C residues in thecysteine-rich signature region as well (FIG. 7B).

Genomic Southern analyses demonstrate that the iGluR of Arabidopsis arebona-fide Arabidopsis genes as shown by the strongly hybridizing DNAfragments in Arabidopsis genomic DNA under high stringency condition(FIG. 8). The weakly hybridizing bands in each lane indicate that theremay be additional GluR genes in the Arabidopsis genome. In theseSouthern blot analyses, two μg of CsC1-purified Arabidopsis genomic DNAwas digested with different restriction enzymes. The digested DNA waselectrophoresed on a 1% (w/v) Tris-phosphate-EDTA agarose gel. The DNAwas transferred to a nylon membrane after depurination, denaturation andneutralization steps. Hybridization steps were carried out under highstringency conditions (65° C. in 0.5×SSC) or low stringency conditions(50° C. in 1×SSC).

Southern blot analyses using the Arabidopsis iGluR cDNAs on genomic DNAfrom other dicots (tobacco), a legume (pea), and two monocots (corn andrice) were performed under low-stringency conditions. This Southern blotanalysis demonstrates that all plant species possess the iGluR genesdescribed for Arabidopsis.

Genomic Southern blot analysis using Arabidopsis mGluR cDNA on genomicDNA from other dicots (tobacco), a legume (pea) and two monocots (cornand rice) were performed under low stringency conditions (50° C. in1×SSC). The analysis demonstrated bands hybridizing to the mGluR in allspecies.

7. EXAMPLE Use of iGluR Agonist (KA) and Antagonist (DNQX) to Define aFunctional iGluR in Arabidopsis

The Arabidopsis iGluR cDNA (pAt-iGR-1) shows high identity to the animalglutamate receptors specifically activated by an iGluR agonist calledkainic acid (KA). These kainate-selective iGluR receptors arecompetitively inhibited by an iGluR antagonist called6,7-dinitroquinoxaline (DNQX). This iGluR agonist (KA) and antagonist(DNQX) are structurally distinct from glutamate (see FIG. 10), yet theybind to the iGluR receptor and stimulate or inhibit its action. Thus,any responses which these drugs may effect in plants, are likely to bedue to their specific interaction with a iGluR-type receptor, ratherthan to general effects caused by inhibition of glutamate-utilizingenzymes. The experiments described below show that agonists, such as KA,mimic the effects of glutamate on gene induction and by contrast, thatantagonists such as DNQX suppress glutamate gene induction. Thus, theseexperiments provide evidence that plants express a functional GluR.

7.1. Effect of GluR Agonists and Antagonists on Plant Growth

A dose-response curve using various concentrations of KA or DNQX wasperformed and the effects of each drug on plant growth was determined bymeasuring root length on vertical tissue culture plates (see FIG. 11).Low concentrations of agonist (KA) have no adverse effects on plantgrowth (FIG. 1A, left panel, compare 0 to 0.3 mM KA). Low concentrationsof DNQX also have no adverse affects on growth (FIG. 11B, left panel,compare 0 to 0.1 mM KA). At high concentrations of agonist (KA) orantagonist (DNQX), a reduction in root growth is observed (FIGS. 11A &B, right panels, black bars). In each case, the effect of high doses ofKA or DNQX is at least partially reversed by glutamate supplementation(FIGS. 12A & B, right panels, white bars). The partial rescue byglutamate indicates that the inhibitory effects of high KA or DNQX arespecific to a glutamate-related process.

7.2. The iGluR Agonist (KA) is Able to Induce the Expression of PlantNitrogen-Assimilatory Genes

The ability of a iGluR agonist to mimic the glutamate-stimulatedexpression of nitrogen assimilatory genes in Arabidopsis was testedusing low concentrations of agonist KA and monitoring ASN1 mRNA levelsas a measure of gene induction by KA. The results demonstrate that thisiGluR agonist pair can specifically affect the expression of nitrogenmetabolic genes in Arabidopsis. These data provide evidence that plantspossess a functional glutamate receptor.

ASN1 mRNA accumulates to high levels specifically in dark-treated plants(FIG. 12A, lane 2), and is repressed by light (FIG. 12A, lane 1) orsucrose (FIG. 12A, lane 3). The results demonstrate that glutamate isable to partially relieve this sucrose repression (FIG. 12A, comparelane 6 to lane 3). Two concentrations of the iGluR agonist KA (300 uMand 30 μM) are each able to relieve the: sucrose repression of ASN1 mRNA(FIG. 12A, lanes 4 & 5). ASN1 mRNA levels-are determined by Northernblot analysis. FIG. 12B shows a quantitative bar graph of these Northernblot results. Quantitatively, KA and glutamate are each able to relievethe sucrose repression of ASN1 mRNA to nearly equivalent levels (FIG.12C). This roughly 2-fold induction of ASN1 mRNA by KA or glutamate iscomparable to the level of metabolic induction of several amino acidgenes in yeast (2-4 fold) (Zalkin, H. and Yanofsky, C., 1982, J. Biol.Chem. 257:1491-1500). Conversely, the iGluR antagonist DNQX seems topartially inhibit glutamate-induction of ASN1 mRNA (FIGS. 12A & B,compare lanes 6 & 7). The discovery that an iGluR agonist can mimicglutamate induction of a plant gene indicates that a functional iGluRglutamate receptor exists in plants.

7.3. Linking the Arabidopsis iGluR Gene to a Functional GlutamateReceptor in Plants

The next experiments demonstrate that the iGluR gene(s) is responsiblefor the observed in vivo responses of ASN1 gene expression to iGluRagonist (KA) or iGluR antagonist (DNQX), using a genetic approach.Arabidopsis mutants are screened for insensitivity or supersensitivityto the iGluR agonist KA or antagonist DNQX. Arabidopsis mutants wereselected in which a mutation in a iGluR gene may improve or disturb thebinding affinity of the agonist or antagonist. In this assay,Arabidopsis seeds are plated on tissue culture plates in Ms Medium(Murashige & Skoog Salt Mixture-plant basic medium available from Gibco(BRL). Agonists and antagonists are added in serial dilution to themedium. The plates are grown vertically in a growth chamber at 22° C.with a 16 hour light/8 hour dark cycle for two weeks. The effect ofagonists and antagonists is determined by changes in root length.Mutagenized M2 Arabidopsis seedlings were screened for those which areinsensitive (resistant) to the iGluR agonist (KA) or antagonist (DNQX)by screening for long roots on high concentrations of either drug (FIG.15A). Conversely, mutants that are super-sensitive to either drug arebeing identified by screening for seedlings with short roots on lowdoses of KA or DNQX (FIG. 14B).

8. EXAMPLE Use of Agonists and Antagonists to Define a Functional mGluRin Arabidopsis

The Arabidopsis mGluR cDNA (pAt-mGR-1) shows high identity to the animalglutamate receptors specifically activated by mGluR agonists calledL-AP4 and ACPD. These mGluR receptors are specifically inhibited bymGluR antagonists L-AP3 (L-(+) amino phosphoropropionate) and L-ABHA(L-aspartate-B-hydroxymate). Any responses which these drugs may effectin plants are likely due to their specific interaction with anmGluR-type receptor, rather than to general effects caused by inhibitionof glutamate-utilizing enzymes. In order to test that plants express afunctional mGluR the assay described herein can be used, whereinagonists such as L-AP4 and ACPD will mimic the effects of glutamate ongene induction and by contrast antagonists such as L-AP3 and L-ABHAsuppress glutamate gene induction. The effects of theseagonists/antagonists can be tested using the vertical root growth assayas described in the working example, Section 7. Serial dilutions of theagonists/antagonists to be tested can range from 3 mM to 0 mM. In eachcase, the effects of doses of L-AP3, L-ABHA, L-AP4 and ACPD should bereversed by glutamate supplementation, indicating that the effect ofthese drugs are specific to glutamate related processes.

9. EXAMPLE The Complete Sequence of the iGluR cDNA

The nucleotide deduced amino acid sequence of the full lengthArabidopsis iGluR cDNA called iGlr1 was determined (five pages long)(FIG. 15). The nucleotide sequence of the full-length Arabidopsis iGlr1cDNA clone (see below) is shown, as is the deduced encoded protein. Theregions of highest homology to animal iGluR are denoted in FIGS. 17 &18. The full-length Arabidopsis iGlr1 cDNA clone was constructed asfollows: the partial EST cDNA clone 107M14T7 was used as a hybridizationprobe to isolate two additional iGlr cDNA clones (HM299 and HM262) fromtwo different Arabidopsis cDNA libraries, KC-HM1 and CD4-7 (obtainedfrom the Arabidopsis stock center, Ohio). Portions of each iGlr cDNAclone were annealed to generate a full-length Arabidopsis iGlr1 cDNAwhich was given the trivial name HM330.

10. EXAMPLE iGluR Antagonist DNQX Phenocopies Plant Mutants Impaired inLight Signal Transduction

This example demonstrates that a specific antagonist of iGluR, DNQX,phenocopies plant mutants impaired in light signal transduction. Lightinduces leaf expansion and chloroplast development (greening) inArabidopsis. Light also inhibits hypocotyl elongation. Plants germinatedin the dark show an etiolated morphology, yellow unopened cotyledons andlong hypocotyl.

Light normally promotes greening and inhibits hypocotyl elongation inwild-type seedlings. hy mutants are impaired in light perception/signaltransduction. hy mutants when grown in light take on the morphology ofdark-grown seedlings (long-hypocotyl) (FIG. 20). When wild-type plantsare treated with DNQX, an iGluR antagonist, they grow as hy mutants(long hypocotyl). Plants grown on normal MS media show a shorthypocotyl, while plants grown on media containing 400 μM DNQX showelongated hypocotyl, similar to hy mutants (FIG. 21).

Plants grown in darkness have unopened yellow cotyledons, but if exposedto light for 5 hrs. the cotyledons begin to green. If plants are grownin the dark with DNQX in the media, the cotyledons remain yellow andunopened even after 5 hrs. of light exposure. Thus DNQX blockslight-induced chloroplast development in Arabidopsis (FIG. 22).

In conclusion, DNQX a specific antagonist of iGlur, inhibits the plant'sresponses to light signal transduction, e.g., induction of leafexpansion and chloroplast development (greening).

11. EXAMPLE iGluR Antagonist DNQX Inhibits Light Induced ChlorophyllSynthesis

This example demonstrates that a specific antagonist of iGluR, DNQX,phenocopies plant mutants impaired in light signal transduction. Lightinduces synthesis of chlorophyll a and chlorophyll b in Arabidopsis, andtreatment with DNQX inhibits this light induced response.

Arabidopsis seedlings were sown on MS+3% sucrose agar plates and grownin complete darkness for 5 days. On day 6, half of the seedlings werekept in the dark (D) and the other half were transferred to white light(L) for 7.5 hours and were grown in the presence or absence of 0.4 mMDNXQ (FIG. 31). Levels of chlorophyll a and chlorophyll b were measuredusing the method of Moran (Moran et al., 1982, Plant Physiol.69:1376-13810). The cotyledons of 30-40 plants were used for each datapoint.

The presence of DNQX significantly inhibits synthesis of chlorophyll aand b in Arabidopsis plants grown in the light. Therefore DNQX, aspecific antagonist of iGluR, inhibits the plant's responses to lightsignal transduction to induce synthesis of chlorophyll.

12. EXAMPLE The iGluR Agonist Kainate (KA) Inhibits Light-InducedGermination

Kainate inhibition of germination in the dark is reversed by glutamate.Plants germinated in the dark on “normal” MS media (MS) as a positivecontrol. Kainate, the iGluR agonist, causes a specific inhibition ofgermination of plants grown in MS media containing KA. The inhibition ofgermination by KA is reversed by the addition of glutamate to the media(MS+KA+Glu) (FIG. 23). Germination involves a light signal. Dark-grownseedlings are sown in the light and transferred to the dark. Theseresults indicate that at high concentrations, the iGluR agonist is ableto block light-induced germination, and that glutamate the naturalagonist for iGluR can reverse the inhibitory effect of high KA. Highdoses of KA an agonist of iGluR in animals functions as a neurotoxin.

The effects of kainate on germination of Arabidopsis in the dark wasdetermined. Arabidopsis seedlings germinated on media containingincreasing amounts of kainate (200-400 uM) show a significant inhibitionof germination of dark-grown seedlings. This inhibition of germinationis likely to be specific to iGluR as it is specifically reversed by thesupplementation of glutamate to the growth media (FIG. 23).

13. EXAMPLE The Arabidopsis iGlr-1 Gene Maps to Chromosome III

The Arabidopsis iGlr-1 gene was mapped using recombinant inbred lines ofArabidopsis. An RFLP for iGlr1 was identified in the wild-typeArabidopsis ecotypes Columbia (C) and Landsberg (L). This iGlr1-specificRFLP was used to identify the genotype of the iGlr1 gene in 30Recombinant Inbred lines as being derived from the C or L parents. The“pattern” of inheritance of the iGlr1 gene in the recombinant inbredlines was compared to known markers and used to determine a map position(see FIG. 24).

The iGlr-1 gene maps to chromosome III to a similar position as twoknown mutants, hy2 and spy (FIG. 25). Using data from recombinant inbredlines hy2 is a mutant impaired in light signal transduction (see reviewWhitelam & Harberd, Plant Cell Environment 1994, 17, 615-625. The spymutant is impaired in GA hormone signal transduction (Jacobsen &Olszewski, 1993, Plant Cell 5, 887-896).

14. EXAMPLE Isolation of Arabdiopsis Mutants Super Sensitive to DNQX

In this assay Arabidopsis mutants were screened for altered sensitivityto the iGluR antagonist DNQX. Mutants affected in iGluR can be used totest the in vivo function of plant iGluR and could also be mappedrelative to the cloned gene. To isolate mutants in Arabidopsis iGluRplants were screened for super-sensitivity to the iGluR antagonist DNQX.Normally wild-type Arabidopsis only show an elongated hypocotylphenotype when exposed to high doses of DNQX (200-400 uM) and show nohypocotyl elongation at low concentrations (100 uM) (see FIG. 21). Theems mutagenized M2 seeds were germinated on media containing 100 uM DNQXand mutants that displayed an elongated hypocotyl at this low dose ofDNQX were selected.

Putative Arabidopsis mutants that are super-sensitive to the iGluRantagonist DNQX were isolated. Arabidopsis wild-type seedlings that showno significant hypocotyl elongation when germinated on 100 uM DNQX werecompared to control MS. By contrast the putative DNQX supersensitivemutant shows an elongated hypocotyl when germinated on 100 uM DNQX. Theputative super-sensitive mutant also demonstrates an elongated hypocotylcompared to wild-type when germinated in the absence of DNQX. Anadditional DNQX supersensitive mutant was isolated that demonstrated ahomeotic phenotype. Lateral roots emerged from the elongation hypocotylof the plant (see FIG. 33). In wild type plants, lateral roots emergeonly from roots.

Thus mutations in iGluR caused a phenotype of light-insensitivity,supporting the Applicants' model.

15. EXAMPLE Arabidopsis Mutants Resistant to Kainate Display a GiantPhenotype

Arabidopsis mutants resistant to the iGluR agonist Kainate wereselected. High doses of kainate (12 mM) kill wild-type seedlings. Thisis not unexpected as high doses of the iGluR agbnist kainate function asa neurotoxin in animals. Arabidopsis mutants with putative defects inthe KA binding site of iGluR were selected by selecting for mutants ableto grow in the presence of 12 mM kainate. The ems mutagenizedArabidopsis M2 seedlings were sown on 12 mM kainate. Note the oneKA-resistant plant is enlarged in Panel B, (FIG. 28).

Arabidopsis mutants resistant to the iGluR agonist kainate, display aGiant phenotype. Two independent Arabidopsis were mutants selected forgrowth on 12 mM KA (see FIG. 28). When the putative KA-resistant plantsare transferred to soil, they each display varying degrees of a Giantvegetative phenotype. This result may indicate that iGluRaffects/enhances overall plant growth.

Two additional independent KA-resistant mutants (KA-giant-1 andKA-giant-2) show a giant, late flowering phenotype (FIG. 32). Some ofthe cauline leaves from the flowering stalks of these KA-resistant giantmutants (panel B) display the morphology of rosette leaves in contrastto the normal morphology in wild type plants (panel A). These resultssuggest that iGluR may be involved in flowering and developmentalprocesses.

A number of references have been cited and the entire disclosure ofwhich are incorporated herein by reference.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are with in the scope of the invention. Indeed variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

36 41 amino acids amino acid <Unknown> unknown peptide 1 Gln Arg Asp LysTyr Asp Ala Ala Val Gly Asp Ile Thr Ile Thr Ser 1 5 10 15 Asn Arg SerLeu Tyr Val Asp Phe Thr Leu Pro Tyr Thr Asp Ile Gly 20 25 30 Ile Gly IleLeu Thr Val Lys Lys Lys 35 40 41 amino acids amino acid <Unknown>unknown peptide 2 Gln Thr Lys Asn Val Asp Leu Ala Leu Ala Gly Ile ThrIle Thr Asp 1 5 10 15 Glu Arg Lys Lys Ala Ile Asp Phe Ser Asp Gly TyrTyr Lys Ser Gly 20 25 30 Leu Leu Val Met Val Lys Ala Asn Asn 35 40 41amino acids amino acid <Unknown> unknown peptide 3 Leu Arg Gln Glu AlaAsp Ile Ala Val Ala Pro Leu Thr Val Thr Ser 1 5 10 15 Ala Arg Glu GluVal Val Ser Phe Thr Thr Pro Phe Leu Gln Thr Gly 20 25 30 Ile Gly Ile LeuLeu Arg Lys Glu Thr 35 40 41 amino acids amino acid <Unknown> unknownpeptide 4 Ile Arg Lys Glu Ala Asp Leu Ala Ile Ala Pro Leu Thr Ile ThrSer 1 5 10 15 Val Arg Glu Asn Ala Ile Ser Phe Thr Lys Pro Phe Met GlnThr Gly 20 25 30 Ile Gly Ile Leu Leu Lys Lys Asp Thr 35 40 41 aminoacids amino acid <Unknown> unknown peptide 5 Val Tyr Gly Arg Ala Asp ValAla Val Ala Pro Leu Thr Ile Thr Leu 1 5 10 15 Val Arg Glu Glu Val IleAsp Phe Ser Lys Pro Phe Met Ser Leu Gly 20 25 30 Ile Ser Ile Met Ile LysLys Pro Gln 35 40 41 amino acids amino acid <Unknown> unknown peptide 6Val Tyr Gly Arg Ala Asp Ile Ala Val Ala Pro Leu Thr Ile Thr Leu 1 5 1015 Val Arg Glu Glu Val Ile Asp Phe Ser Lys Pro Phe Met Ser Leu Gly 20 2530 Ile Ser Ile Met Ile Lys Lys Pro Gln 35 40 41 amino acids amino acid<Unknown> unknown peptide 7 Val Tyr Gly Lys Ala Asp Ile Ala Ile Ala ProLeu Thr Ile Thr Leu 1 5 10 15 Val Arg Glu Glu Val Ile Asp Phe Ser LysPro Phe Met Ser Leu Gly 20 25 30 Ile Ser Ile Met Ile Lys Lys Pro Gln 3540 262 amino acids amino acid <Unknown> unknown protein 8 Arg Gly AsnAsn Asp Asn Leu Ala Tyr Leu Leu Ser Thr Gln Arg Asp 1 5 10 15 Lys TyrAsp Ala Ala Val Gly Asp Ile Thr Ile Thr Ser Asn Arg Ser 20 25 30 Leu TyrVal Asp Phe Thr Leu Pro Tyr Thr Asp Ile Gly Ile Gly Ile 35 40 45 Leu ThrVal Lys Lys Lys Ser Gln Gly Met Trp Thr Phe Phe Asp Pro 50 55 60 Phe GluLys Ser Leu Trp Leu Ala Ser Gly Ala Phe Phe Val Leu Thr 65 70 75 80 GlyIle Val Val Trp Leu Val Glu Arg Pro Val Asn Pro Glu Phe Gln 85 90 95 GlySer Trp Gly Gln Gln Leu Ser Met Met Leu Leu Val Trp Ile Leu 100 105 110Leu Pro Leu Cys Leu Leu Thr Gly Glu Lys Leu Gln Lys Met Ser Ser 115 120125 Arg Phe Leu Val Ile Val Trp Val Phe Val Val Leu Ile Leu Thr Ser 130135 140 Ser Tyr Ser Ala Asn Leu Thr Ser Thr Lys Thr Ile Ser Arg Met Gln145 150 155 160 Leu Asn His Gln Met Val Phe Gly Gly Ser Thr Thr Ser MetThr Ala 165 170 175 Lys Leu Gly Ser Ile Asn Gly Gly Gly Gly Leu Cys ThrThr Leu Arg 180 185 190 Asp Gly Thr Leu Thr His Val Ile Asn Glu Ile ProTyr Leu Ser Ile 195 200 205 Leu Ile Gly Asn Tyr Pro Asn Asp Phe Val MetThr Asp Arg Val Thr 210 215 220 Asn Thr Asn Gly Phe Gly Phe Met Phe GlnLys Gly Ser Asp Leu Val 225 230 235 240 Pro Lys Val Ser Arg Glu Ile AlaLys Leu Arg Ser Leu Gly Met Leu 245 250 255 Lys Asp Met Glu Glu Lys 260295 amino acids amino acid <Unknown> unknown protein 9 Tyr Leu Val ThrAsn Gly Lys His Gly Lys Lys Val Asn Asn Val Trp 1 5 10 15 Asn Gly MetIle Gly Glu Val Val Tyr Gln Arg Ala Val Met Ala Val 20 25 30 Gly Ser LeuThr Ile Asn Glu Glu Arg Ser Glu Val Val Asp Phe Ser 35 40 45 Val Pro PheVal Glu Thr Gly Ile Ser Val Met Val Ser Arg Ser Asn 50 55 60 Gly Thr ValSer Pro Ser Ala Phe Leu Glu Pro Phe Ser Ala Ser Val 65 70 75 80 Trp ValMet Met Phe Val Met Leu Leu Ile Val Ser Ala Ile Ala Val 85 90 95 Phe ValPhe Glu Tyr Phe Ser Pro Val Gly Tyr Asn Arg Asn Leu Ala 100 105 110 LysGly Lys Ala Pro His Gly Pro Ser Phe Thr Ile Gly Lys Ala Ile 115 120 125Trp Leu Leu Trp Gly Leu Val Phe Asn Asn Ser Val Pro Val Gln Asn 130 135140 Pro Lys Gly Thr Thr Ser Lys Ile Met Val Ser Val Trp Ala Phe Phe 145150 155 160 Ala Val Ile Phe Leu Ala Ser Tyr Thr Ala Asn Leu Ala Ala PheMet 165 170 175 Ile Gln Glu Glu Phe Val Asp Gln Val Thr Gly Leu Ser AspLys Lys 180 185 190 Phe Gln Arg Pro His Asp Tyr Ser Pro Pro Phe Arg PheGly Thr Val 195 200 205 Pro Asn Gly Ser Thr Glu Arg Asn Ile Arg Asn AsnTyr Pro Tyr Met 210 215 220 His Gln Tyr Met Thr Lys Phe Asn Gln Lys GlyVal Glu Asp Ala Leu 225 230 235 240 Val Ser Leu Lys Thr Gly Lys Leu AspAla Phe Ile Tyr Asp Ala Ala 245 250 255 Val Leu Asn Tyr Lys Ala Gly ArgAsp Glu Gly Cys Lys Leu Val Thr 260 265 270 Ile Gly Ser Gly Tyr Ile PheAla Thr Thr Gly Tyr Gly Ile Ala Leu 275 280 285 Gln Lys Gly Ser Pro TrpLys 290 295 297 amino acids amino acid <Unknown> unknown protein 10 SerTyr Glu Ile Arg Leu Val Glu Asp Gly Lys Tyr Gly Ala Gln Asp 1 5 10 15Asp Lys Gly Gln Trp Asn Gly Met Val Lys Glu Leu Ile Asp His Lys 20 25 30Ala Asp Leu Ala Val Ala Pro Leu Thr Ile Thr His Val Arg Glu Lys 35 40 45Ala Ile Asp Phe Ser Lys Pro Phe Met Thr Leu Gly Val Ser Ile Leu 50 55 60Tyr Arg Lys Pro Asn Gly Thr Asn Pro Ser Val Phe Ser Phe Leu Asn 65 70 7580 Pro Leu Ser Pro Asp Ile Trp Met Tyr Val Leu Leu Ala Tyr Leu Gly 85 9095 Val Ser Cys Val Leu Phe Val Ile Ala Arg Phe Ser Pro Tyr Glu Trp 100105 110 Tyr Asp Ala His Pro Cys Asn Pro Gly Ser Glu Val Val Glu Asn Asn115 120 125 Phe Thr Leu Leu Asn Ser Phe Trp Phe Gly Met Gly Ser Leu MetGln 130 135 140 Gln Gly Ser Glu Leu Met Pro Lys Ala Leu Ser Thr Arg IleIle Gly 145 150 155 160 Gly Ile Trp Trp Phe Phe Thr Leu Ile Ile Ile SerSer Tyr Thr Ala 165 170 175 Asn Leu Ala Ala Phe Leu Thr Val Glu Arg MetGlu Ser Pro Ile Asp 180 185 190 Ser Ala Asp Asp Leu Ala Lys Gln Thr LysIle Glu Tyr Gly Ala Val 195 200 205 Lys Asp Gly Ala Thr Met Thr Phe PheLys Lys Ser Lys Ile Ser Thr 210 215 220 Phe Glu Lys Met Trp Ala Phe MetSer Ser Lys Pro Ser Ala Leu Val 225 230 235 240 Lys Asn Asn Glu Glu GlyIle Gln Arg Thr Leu Thr Ala Asp Tyr Ala 245 250 255 Leu Leu Met Glu SerThr Thr Ile Glu Tyr Ile Thr Gln Arg Asn Cys 260 265 270 Asn Leu Thr GlnIle Gly Gly Leu Ile Asp Ser Lys Gly Tyr Gly Ile 275 280 285 Gly Thr ProMet Gly Ser Pro Tyr Arg 290 295 179 amino acids amino acid <Unknown>unknown protein 11 Gly Lys Gly Val Arg Glu Ile Pro Ser Ser Val Cys ThrLeu Pro Cys 1 5 10 15 Lys Pro Gly Gln Arg Lys Lys Thr Gln Lys Gly ThrPro Cys Cys Trp 20 25 30 Thr Cys Glu Pro Cys Asp Gly Tyr Gln Tyr Gln PheAsp Glu Met Thr 35 40 45 Cys Gln His Cys Pro Tyr Asp Gln Arg Pro Asn GluAsn Arg Thr Gly 50 55 60 Cys Gln Asn Ile Pro Ile Ile Lys Leu Glu Trp HisSer Pro Trp Ala 65 70 75 80 Val Ile Pro Val Phe Leu Ala Met Leu Gly IleIle Ala Thr Ile Phe 85 90 95 Val Met Ala Thr Phe Ile Arg Tyr Asn Asp ThrPro Ile Val Arg Ala 100 105 110 Ser Gly Arg Glu Leu Ser Tyr Val Leu LeuThr Gly Ile Phe Leu Cys 115 120 125 Tyr Ile Ile Thr Phe Leu Met Ile AlaLys Pro Asp Val Ala Val Cys 130 135 140 Ser Phe Arg Arg Val Phe Leu GlyLeu Gly Met Cys Ile Ser Tyr Ala 145 150 155 160 Ala Leu Leu Thr Lys ThrAsn Arg Ile Tyr Arg Ile Phe Glu Gln Gly 165 170 175 Lys Lys Ser 115amino acids amino acid <Unknown> unknown peptide 12 Thr Phe Xaa Cys TrpLeu Lys Asn Ala Phe Cys Ala Ser Ser Phe Phe 1 5 10 15 Gln Leu Ser SerMet Glu Pro Tyr Arg Leu Arg Leu Arg Phe Ser Phe 20 25 30 Gln Lys Cys SerIle Ala Ala Phe Leu Gly Pro Ala Val Ser Phe Asn 35 40 45 Ser Ile Glu ArgPhe Leu Asn Ser Leu Ser Thr Ser Leu Ile Phe Val 50 55 60 Xaa Phe Ser SerMet Tyr Phe Leu Ser Xaa Thr Cys Ser Ser Ser Ile 65 70 75 80 Ile Phe SerVal Xaa Val Ile Thr Gly Ala Phe Leu Ala Arg Pro Ser 85 90 95 Ala Pro IleSer Ala Phe Ser Phe Gly Ser Asp Ala Ile Ile Ser Phe 100 105 110 Ser LeuLys 115 235 base pairs nucleic acid single unknown DNA 13 TGAAGATGCAGGACAGGTTC AATGGAGGTA TGATAACCCT CCAGACTTCA ATAGTGTGAA 60 CCAGCTCTTTGAAGAAGGCC AGACTAAGGT GTGGCCAGAA GGTTCGTTAG AAGAGACAG 120 GCAAAACGCGATCAAGTCAT GGGAGATGGA GTTCTCACAT AAGATCCGTT TACAGGACT 180 CAAGACTATAAACCCTGAGA AGTTTAAGCT CTTTTGTCAA TGGGAGAGAA GGTTT 235 177 base pairsnucleic acid single unknown DNA 14 GGTGAATCTT TCGAGGTTGA GGAGGCGGTGGCTCTCGAGT CACAAACCAT AGCGCATATG 60 GTTGAAGACG ACTGCGTNAN CAACGGAGTCCCTCTTCCTA ACGTCACGAG CAAGATCCT 120 GCCAAGGTGA TCGAGTATTG CAAGAGGCACGTCGAGGCTG CTGCCTNTAA AGGCCGA 177 247 base pairs nucleic acid singleunknown DNA 15 TCGTTTGCTC GAAGATCCGC TGCTTGATCT GCTCGCCACA CGCTATNGGAGAGGNAANGG 60 TTAGGGTTAC TNATTTTCCG TCGAGTAGTC TNACNNAAAA CTGCAACGGCTTACAACTT 120 GATCCGCCAT CGATTTTCGA TTCTAAAGCT TGGACGAAGN AGAAGNANAAAGTTCGATT 180 GATTTCTGGA GAGAAATTGG GGGAAAGTTT AAAAACGGAT CCCTAAGGTAGTCTGAGTC 240 CTCTCTC 247 29 amino acids amino acid single linearpeptide 16 Val Arg Pro Asp Pro Glu Thr Gly Val Asn Thr Val Ser Gly PheCys 1 5 10 15 Val Glu Val Phe Lys Thr Cys Ile Ala Pro Phe Asn Tyr 20 2559 amino acids amino acid single linear peptide 17 Glu Leu Glu Phe IlePro Tyr Arg Gly Asn Asn Asp Asn Leu Ala Tyr 1 5 10 15 Leu Leu Ser ThrGln Arg Asp Lys Tyr Asp Ala Ala Val Gly Asp Ile 20 25 30 Thr Ile Thr SerAsn Arg Ser Leu Tyr Val Asp Phe Thr Leu Pro Tyr 35 40 45 Thr Asp Ile GlyIle Gly Ile Leu Thr Val Lys 50 55 37 amino acids amino acid singlelinear peptide 18 Lys Lys Ser Gln Gly Met Trp Thr Phe Phe Asp Pro PheGlu Lys Ser 1 5 10 15 Leu Trp Leu Ala Ser Gly Ala Phe Phe Val Leu ThrGly Ile Val Val 20 25 30 Trp Leu Val Glu Arg 35 16 amino acids aminoacid single linear peptide 19 Ser Val Asn Pro Glu Phe Gln Gly Ser TrpGly Gln Gln Leu Ser Met 1 5 10 15 15 amino acids amino acid singlelinear peptide 20 Met Leu Trp Phe Gly Phe Ser Thr Ile Val Phe Ala HisArg Glu 1 5 10 15 38 amino acids amino acid single linear peptide 21 LysLeu Gln Lys Met Ser Ser Arg Phe Leu Val Ile Val Trp Val Phe 1 5 10 15Val Val Leu Ile Leu Thr Ser Ser Tyr Ser Ala Asn Leu Thr Ser Thr 20 25 30Lys Thr Ile Ser Arg Met 35 5 amino acids amino acid single linearpeptide 22 Gln Leu Asn His Gln 1 5 27 amino acids amino acid singlelinear peptide 23 Met Val Phe Gly Gly Ser Thr Thr Ser Met Thr Ala LysLeu Gly Ser 1 5 10 15 Ile Asn Ala Val Glu Ala Tyr Ala Gln Leu Leu 20 2535 amino acids amino acid single linear peptide 24 Arg Asp Gly Thr LeuAsn His Val Ile Asn Glu Ile Pro Tyr Leu Ser 1 5 10 15 Ile Leu Ile GlyAsn Tyr Pro Asn Asp Phe Val Met Thr Asp Arg Val 20 25 30 Thr Asn Thr 3590 amino acids amino acid single linear peptide 25 Asn Gly Phe Gly PheMet Phe Gln Lys Gly Ser Asp Leu Val Pro Lys 1 5 10 15 Val Ser Arg GluIle Ala Lys Leu Arg Ser Leu Gly Met Leu Lys Asp 20 25 30 Met Glu Lys LysTrp Phe Gln Lys Leu Asp Ser Leu Asn Val His Ser 35 40 45 Asn Thr Glu GluVal Ala Ser Thr Asn Asp Asp Asp Glu Ala Ser Lys 50 55 60 Arg Phe Thr PheArg Glu Leu Arg Gly Leu Phe Ile Ile Ala Gly Ala 65 70 75 80 Ala His ValLeu Val Leu Ala Leu His Leu 85 90 10 amino acids amino acid singlelinear peptide 26 Phe His Thr Arg Gln Glu Val Ser Arg Leu 1 5 10 9 aminoacids amino acid single linear peptide 27 Cys Thr Lys Leu Gln Ser PheTyr Lys 1 5 41 amino acids amino acid single linear peptide 28 Ala AlaGlu Ile Ala Lys His Cys Gly Phe Lys Tyr Lys Leu Thr Ile 1 5 10 15 ValGly Asp Gly Lys Tyr Gly Ala Arg Asp Ala Asp Thr Lys Ile Trp 20 25 30 AsnGly Met Val Gly Glu Leu Val Tyr 35 40 39 amino acids amino acid singlelinear peptide 29 Gly Lys Ala Asp Ile Ala Ile Ala Pro Leu Thr Ile ThrLeu Val Arg 1 5 10 15 Glu Glu Val Ile Asp Phe Ser Lys Pro Phe Met SerLeu Gly Ile Ser 20 25 30 Ile Met Ile Lys Lys Pro Gln 35 259 amino acidsamino acid single linear peptide 30 Lys Ser Lys Pro Gly Val Phe Ser PheLeu Asp Pro Leu Ala Tyr Glu 1 5 10 15 Ile Trp Met Cys Ile Val Phe AlaTyr Ile Gly Val Ser Val Val Leu 20 25 30 Phe Leu Val Ser Arg Phe Ser ProTyr Glu Trp His Thr Glu Glu Phe 35 40 45 Glu Asp Gly Arg Glu Thr Gln SerSer Glu Ser Thr Asn Glu Phe Gly 50 55 60 Ile Phe Asn Ser Leu Trp Phe SerLeu Gly Ala Phe Met Arg Gln Gly 65 70 75 80 Cys Asp Ile Ser Pro Arg SerLeu Ser Gly Arg Ile Val Gly Gly Val 85 90 95 Trp Trp Phe Phe Thr Leu IleIle Ile Ser Ser Tyr Thr Ala Asn Leu 100 105 110 Ala Ala Phe Leu Thr ValGlu Arg Met Val Ser Pro Ile Glu Ser Ala 115 120 125 Glu Asp Leu Ser LysGln Thr Glu Ile Ala Tyr Gly Thr Leu Asp Ser 130 135 140 Gly Ser Thr LysGlu Phe Phe Arg Arg Ser Lys Ile Ala Val Phe Asp 145 150 155 160 Lys MetTrp Thr Tyr Met Arg Ser Ala Glu Pro Ser Val Phe Val Arg 165 170 175 ThrThr Ala Glu Gly Val Ala Arg Val Arg Lys Ser Lys Gly Lys Tyr 180 185 190Ala Tyr Leu Leu Glu Ser Thr Met Asn Glu Tyr Ile Glu Gln Arg Lys 195 200205 Pro Cys Asp Thr Met Lys Val Gly Gly Asn Leu Asp Ser Lys Gly Tyr 210215 220 Gly Ile Ala Thr Pro Lys Gly Ser Ser Leu Gly Thr Pro Val Asn Leu225 230 235 240 Ala Val Leu Lys Leu Ser Glu Gln Gly Val Leu Asp Lys LeuLys Asn 245 250 255 Lys Trp Trp 94 amino acids amino acid single linearpeptide 31 Tyr Asp Lys Gly Glu Cys Gly Ala Lys Asp Ser Gly Ser Lys GluLys 1 5 10 15 Thr Ser Ala Leu Ser Leu Ser Asn Val Ala Gly Val Phe TyrIle Leu 20 25 30 Val Gly Gly Leu Gly Ala Met Leu Val Ala Leu Ile Glu PheCys Tyr 35 40 45 Lys Ser Arg Ala Glu Ala Lys Arg Met Lys Val Ala Lys AsnPro Gln 50 55 60 Asn Ile Asn Pro Ser Ser Ser Gln Asn Ser Gln Asn Phe AlaThr Tyr 65 70 75 80 Lys Glu Gly Tyr Asn Val Tyr Gly Ile Glu Ser Val LysIle 85 90 2484 base pairs nucleic acid single linear mat_peptide1...2424 32 ATG GAG ATT CTG TTT TCT ATT TCC ATT CTT GCT CTT CTC TTT TCCGGA 48 Met Glu Ile Leu Phe Ser Ile Ser Ile Leu Ala Leu Leu Phe Ser Gly 15 10 15 GTA GTA GCT GCT CCA AGC GAC GAT GAT GTT TTC GAA GAG GTT AGG GTT96 Val Val Ala Ala Pro Ser Asp Asp Asp Val Phe Glu Glu Val Arg Val 20 2530 GGA TTG GTG GTT GAC TTG AGT TCT ATT CAA GGC AAG ATT CTG GAA ACT 144Gly Leu Val Val Asp Leu Ser Ser Ile Gln Gly Lys Ile Leu Glu Thr 35 40 45TCT TTT AAC TTA GCG CTT TCA GAT TTC TAT GGC ATC AAC AAT GGA TAC 192 SerPhe Asn Leu Ala Leu Ser Asp Phe Tyr Gly Ile Asn Asn Gly Tyr 50 55 60 CGAACC AGA GTC TCT GTT TTG GTC AGA GAC TCC CAA GGA GAC CCG ATC 240 Arg ThrArg Val Ser Val Leu Val Arg Asp Ser Gln Gly Asp Pro Ile 65 70 75 80 ATTGCT CTT GCC GCC GCT ACT GAT CTT CTC AAA AAT GCA AAA GCG GAA 288 Ile AlaLeu Ala Ala Ala Thr Asp Leu Leu Lys Asn Ala Lys Ala Glu 85 90 95 GCC ATTGTT GGT GCA CAA TCA TTA CAA GAG GCA AAG CTT TTG GCG ACG 336 Ala Ile ValGly Ala Gln Ser Leu Gln Glu Ala Lys Leu Leu Ala Thr 100 105 110 ATT AGCGAA AAA GCT AAA GTT CCG GTC ATA TCT ACT TTC TTG CCA AAC 384 Ile Ser GluLys Ala Lys Val Pro Val Ile Ser Thr Phe Leu Pro Asn 115 120 125 ACG TTATCT TTG AAG AAA TAC GAT AAC TTT ATT CAA TGG ACG CAT GAT 432 Thr Leu SerLeu Lys Lys Tyr Asp Asn Phe Ile Gln Trp Thr His Asp 130 135 140 ACT ACATCA GAG GCT AAG GGA ATT ACA AGT CTC ATA CAA GAT TTC AGT 480 Thr Thr SerGlu Ala Lys Gly Ile Thr Ser Leu Ile Gln Asp Phe Ser 145 150 155 160 TGTAAA TCG GTT GTG GTT ATA TAC GAG GAT GCT GAT GAT TGG AGT GAG 528 Cys LysSer Val Val Val Ile Tyr Glu Asp Ala Asp Asp Trp Ser Glu 165 170 175 AGTTTG CAA ATA TTG GTT GAG AAT TTT CAA GAT AAA GGA ATC TAT ATC 576 Ser LeuGln Ile Leu Val Glu Asn Phe Gln Asp Lys Gly Ile Tyr Ile 180 185 190 GCTCGT TCT GCT TCT TTT GCA GTC TCA TCA TCA GGA GAA AAT CAT ATG 624 Ala ArgSer Ala Ser Phe Ala Val Ser Ser Ser Gly Glu Asn His Met 195 200 205 ATGAAT CAG CTA AGG AAG CTT AAG GTC TCA AGA GCA TCG GTT TTT GTG 672 Met AsnGln Leu Arg Lys Leu Lys Val Ser Arg Ala Ser Val Phe Val 210 215 220 GTGCAT ATG TCC GAG ATT CTT GTT TCT CGT CTC TTC CAA TGT GTA GAG 720 Val HisMet Ser Glu Ile Leu Val Ser Arg Leu Phe Gln Cys Val Glu 225 230 235 240AAG TTA GGT TTG ATG GAA GAA GCG TTC GCT TGG ATC CTC ACT GCA AGA 768 LysLeu Gly Leu Met Glu Glu Ala Phe Ala Trp Ile Leu Thr Ala Arg 245 250 255ACC ATG AAC TAC TTG GAA CAT TTT GCA ATA ACT AGG TCG ATG CAA GGG 816 ThrMet Asn Tyr Leu Glu His Phe Ala Ile Thr Arg Ser Met Gln Gly 260 265 270GTC ATT GGT TTC AAA TCT TAC ATC CCT GTA TCT GAA GAA GTT AAG AAT 864 ValIle Gly Phe Lys Ser Tyr Ile Pro Val Ser Glu Glu Val Lys Asn 275 280 285TTT ACT TCA AGA TTG AGG AAA CGT ATG GGA GAT GAT ACA GAA ACA GAG 912 PheThr Ser Arg Leu Arg Lys Arg Met Gly Asp Asp Thr Glu Thr Glu 290 295 300CAT TCT AGT GTA ATC ATC GGT TTA CGC GCA CAC GAT ATC GCT TGT ATT 960 HisSer Ser Val Ile Ile Gly Leu Arg Ala His Asp Ile Ala Cys Ile 305 310 315320 CTA GCA AAT GCA GTA GAG AAG TTC AGT GTA AGT GGT AAA GTT GAA GCA 1008Leu Ala Asn Ala Val Glu Lys Phe Ser Val Ser Gly Lys Val Glu Ala 325 330335 TCT TCG AAT GTA TCA GCT GAT CTT CTG GAT ACA ATT AGA CAT AGT AGA 1056Ser Ser Asn Val Ser Ala Asp Leu Leu Asp Thr Ile Arg His Ser Arg 340 345350 TTC AAG GGT TTG AGT GGT GAC ATC CAA ATC TCT GAC AAC AAA TTT ATC 1104Phe Lys Gly Leu Ser Gly Asp Ile Gln Ile Ser Asp Asn Lys Phe Ile 355 360365 TCA GAG ACA TTT GAA ATC GTG AAT ATT GGA AGA GAA AAA CAG AGA AGG 1152Ser Glu Thr Phe Glu Ile Val Asn Ile Gly Arg Glu Lys Gln Arg Arg 370 375380 ATA GGA TTA TGG AGT GGT GGT AGT TTT AGC CAA AGA AGA CAG ATT GTT 1200Ile Gly Leu Trp Ser Gly Gly Ser Phe Ser Gln Arg Arg Gln Ile Val 385 390395 400 TGG CCT GGC AGG TCT CGT AAG ATC CCA AGA CAC CGT GTT TTG GCA GAG1248 Trp Pro Gly Arg Ser Arg Lys Ile Pro Arg His Arg Val Leu Ala Glu 405410 415 AAA GGT GAA AAG AAG GTG CTT AGG GTC TTA GTT ACC GCA GGA AAC AAG1296 Lys Gly Glu Lys Lys Val Leu Arg Val Leu Val Thr Ala Gly Asn Lys 420425 430 GTC CCG CAT CTA GTG TCG GTG CGT CCT GAT CCT GAA ACA GGT GTT AAT1344 Val Pro His Leu Val Ser Val Arg Pro Asp Pro Glu Thr Gly Val Asn 435440 445 ACT GTC TCT GGA TTC TGC GTA GAG GTT TTC AAG ACT TGC ATT GCT CCT1392 Thr Val Ser Gly Phe Cys Val Glu Val Phe Lys Thr Cys Ile Ala Pro 450455 460 TTT AAC TAC GAG CTT GAA TTC ATA CCT TAC CGT GGA AAC AAT GAC AAT1440 Phe Asn Tyr Glu Leu Glu Phe Ile Pro Tyr Arg Gly Asn Asn Asp Asn 465470 475 480 CTT GCT TAT CTA CTT TCT ACT CAG AGA GAC AAG TAT GAT GCA GCAGTT 1488 Leu Ala Tyr Leu Leu Ser Thr Gln Arg Asp Lys Tyr Asp Ala Ala Val485 490 495 GGT GAT ATC ACC ATC ACT TCC AAC AGA TCT TTG TAT GTT GAT TTTACT 1536 Gly Asp Ile Thr Ile Thr Ser Asn Arg Ser Leu Tyr Val Asp Phe Thr500 505 510 TTG CCG TAC ACT GAC ATT GGT ATT GGA ATC CTG ACA GTA AAA AAGAAA 1584 Leu Pro Tyr Thr Asp Ile Gly Ile Gly Ile Leu Thr Val Lys Lys Lys515 520 525 AGC CAA GGG ATG TGG ACT TTC TTT GAT CCT TTT GAA AAA TCC TTGTGG 1632 Ser Gln Gly Met Trp Thr Phe Phe Asp Pro Phe Glu Lys Ser Leu Trp530 535 540 CTA GCA AGT GGA GCT TTC TTT GTC TTA ACT GGG ATT GTT GTT TGGTTA 1680 Leu Ala Ser Gly Ala Phe Phe Val Leu Thr Gly Ile Val Val Trp Leu545 550 555 560 GTT GAA CGG TCC GTT AAT CCG GAA TTT CAG GGC TCT TGG GGACAA CAA 1728 Val Glu Arg Ser Val Asn Pro Glu Phe Gln Gly Ser Trp Gly GlnGln 565 570 575 CTT AGT ATG ATG CTC TGG TTT GGT TTC TCA ACC ATT GTA TTTGCT CAC 1776 Leu Ser Met Met Leu Trp Phe Gly Phe Ser Thr Ile Val Phe AlaHis 580 585 590 AGA GAG AAG CTA CAG AAA ATG TCA TCA AGA TTC TTA GTC ATAGTT TGG 1824 Arg Glu Lys Leu Gln Lys Met Ser Ser Arg Phe Leu Val Ile ValTrp 595 600 605 GTT TTT GTG GTG TTA ATA TTG ACT TCA AGT TAC AGC GCA AACTTG ACA 1872 Val Phe Val Val Leu Ile Leu Thr Ser Ser Tyr Ser Ala Asn LeuThr 610 615 620 TCA ACC AAG ACC ATT TCT CGC ATG CAA TTA AAT CAT CAG ATGGTT TTC 1920 Ser Thr Lys Thr Ile Ser Arg Met Gln Leu Asn His Gln Met ValPhe 625 630 635 640 GGG GGA TCT ACG ACG TCA ATG ACT GCG AAG CTC GGA TCCATT AAT GCA 1968 Gly Gly Ser Thr Thr Ser Met Thr Ala Lys Leu Gly Ser IleAsn Ala 645 650 655 GTT GAG GCC TAT GCA CAA CTT TTG CGA GAT GGA ACT CTTAAT CAT GTC 2016 Val Glu Ala Tyr Ala Gln Leu Leu Arg Asp Gly Thr Leu AsnHis Val 660 665 670 ATC AAT GAA ATA CCT TAT CTC AGT ATC CTT ATC GGA AATTAT CCG AAT 2064 Ile Asn Glu Ile Pro Tyr Leu Ser Ile Leu Ile Gly Asn TyrPro Asn 675 680 685 GAT TTC GTA ATG ACA GAT AGA GTG ACT AAT ACC AAT GGCTTT GGC TTT 2112 Asp Phe Val Met Thr Asp Arg Val Thr Asn Thr Asn Gly PheGly Phe 690 695 700 ATG TTC CAG AAA GGT TCG GAT TTG GTT CCT AAA GTA TCGCGA GAA ATC 2160 Met Phe Gln Lys Gly Ser Asp Leu Val Pro Lys Val Ser ArgGlu Ile 705 710 715 720 GCG AAG CTA AGA TCA TTG GGA ATG TTG AAA GAC ATGGAG AAA AAA TGG 2208 Ala Lys Leu Arg Ser Leu Gly Met Leu Lys Asp Met GluLys Lys Trp 725 730 735 TTT CAA AAA CTG GAT TCA CTA AAT GTA CAT TCC AACACC GAG GAA GTT 2256 Phe Gln Lys Leu Asp Ser Leu Asn Val His Ser Asn ThrGlu Glu Val 740 745 750 GCA TCT ACC AAC GAC GAT GAT GAG GCA TCT AAG CGATTC ACC TTC CGT 2304 Ala Ser Thr Asn Asp Asp Asp Glu Ala Ser Lys Arg PheThr Phe Arg 755 760 765 GAG TTG CGC GGT TTG TTC ATC ATT GCG GGA GCT GCTCAT GTT CTC GTA 2352 Glu Leu Arg Gly Leu Phe Ile Ile Ala Gly Ala Ala HisVal Leu Val 770 775 780 CTA GCC CTA CAT CTC TTT CAT ACG CGT CAA GAG GTATCA CGA CTA TGC 2400 Leu Ala Leu His Leu Phe His Thr Arg Gln Glu Val SerArg Leu Cys 785 790 795 800 ACC AAA CTT CAA AGC TTC TAT AAG TAA AAA GTGATC CAT CGT TCA TAA 2448 Thr Lys Leu Gln Ser Phe Tyr Lys * Lys Val IleHis Arg Ser * 805 810 815 GCT CTA CTA TAG CAA TTG ACG GGA CAG GAC TCATAA 2484 Ala Leu Leu * Gln Leu Thr Gly Gln Asp Ser * 820 825 808 aminoacids amino acid single linear protein 33 Met Glu Ile Leu Phe Ser IleSer Ile Leu Ala Leu Leu Phe Ser Gly 1 5 10 15 Val Val Ala Ala Pro SerAsp Asp Asp Val Phe Glu Glu Val Arg Val 20 25 30 Gly Leu Val Val Asp LeuSer Ser Ile Gln Gly Lys Ile Leu Glu Thr 35 40 45 Ser Phe Asn Leu Ala LeuSer Asp Phe Tyr Gly Ile Asn Asn Gly Tyr 50 55 60 Arg Thr Arg Val Ser ValLeu Val Arg Asp Ser Gln Gly Asp Pro Ile 65 70 75 80 Ile Ala Leu Ala AlaAla Thr Asp Leu Leu Lys Asn Ala Lys Ala Glu 85 90 95 Ala Ile Val Gly AlaGln Ser Leu Gln Glu Ala Lys Leu Leu Ala Thr 100 105 110 Ile Ser Glu LysAla Lys Val Pro Val Ile Ser Thr Phe Leu Pro Asn 115 120 125 Thr Leu SerLeu Lys Lys Tyr Asp Asn Phe Ile Gln Trp Thr His Asp 130 135 140 Thr ThrSer Glu Ala Lys Gly Ile Thr Ser Leu Ile Gln Asp Phe Ser 145 150 155 160Cys Lys Ser Val Val Val Ile Tyr Glu Asp Ala Asp Asp Trp Ser Glu 165 170175 Ser Leu Gln Ile Leu Val Glu Asn Phe Gln Asp Lys Gly Ile Tyr Ile 180185 190 Ala Arg Ser Ala Ser Phe Ala Val Ser Ser Ser Gly Glu Asn His Met195 200 205 Met Asn Gln Leu Arg Lys Leu Lys Val Ser Arg Ala Ser Val PheVal 210 215 220 Val His Met Ser Glu Ile Leu Val Ser Arg Leu Phe Gln CysVal Glu 225 230 235 240 Lys Leu Gly Leu Met Glu Glu Ala Phe Ala Trp IleLeu Thr Ala Arg 245 250 255 Thr Met Asn Tyr Leu Glu His Phe Ala Ile ThrArg Ser Met Gln Gly 260 265 270 Val Ile Gly Phe Lys Ser Tyr Ile Pro ValSer Glu Glu Val Lys Asn 275 280 285 Phe Thr Ser Arg Leu Arg Lys Arg MetGly Asp Asp Thr Glu Thr Glu 290 295 300 His Ser Ser Val Ile Ile Gly LeuArg Ala His Asp Ile Ala Cys Ile 305 310 315 320 Leu Ala Asn Ala Val GluLys Phe Ser Val Ser Gly Lys Val Glu Ala 325 330 335 Ser Ser Asn Val SerAla Asp Leu Leu Asp Thr Ile Arg His Ser Arg 340 345 350 Phe Lys Gly LeuSer Gly Asp Ile Gln Ile Ser Asp Asn Lys Phe Ile 355 360 365 Ser Glu ThrPhe Glu Ile Val Asn Ile Gly Arg Glu Lys Gln Arg Arg 370 375 380 Ile GlyLeu Trp Ser Gly Gly Ser Phe Ser Gln Arg Arg Gln Ile Val 385 390 395 400Trp Pro Gly Arg Ser Arg Lys Ile Pro Arg His Arg Val Leu Ala Glu 405 410415 Lys Gly Glu Lys Lys Val Leu Arg Val Leu Val Thr Ala Gly Asn Lys 420425 430 Val Pro His Leu Val Ser Val Arg Pro Asp Pro Glu Thr Gly Val Asn435 440 445 Thr Val Ser Gly Phe Cys Val Glu Val Phe Lys Thr Cys Ile AlaPro 450 455 460 Phe Asn Tyr Glu Leu Glu Phe Ile Pro Tyr Arg Gly Asn AsnAsp Asn 465 470 475 480 Leu Ala Tyr Leu Leu Ser Thr Gln Arg Asp Lys TyrAsp Ala Ala Val 485 490 495 Gly Asp Ile Thr Ile Thr Ser Asn Arg Ser LeuTyr Val Asp Phe Thr 500 505 510 Leu Pro Tyr Thr Asp Ile Gly Ile Gly IleLeu Thr Val Lys Lys Lys 515 520 525 Ser Gln Gly Met Trp Thr Phe Phe AspPro Phe Glu Lys Ser Leu Trp 530 535 540 Leu Ala Ser Gly Ala Phe Phe ValLeu Thr Gly Ile Val Val Trp Leu 545 550 555 560 Val Glu Arg Ser Val AsnPro Glu Phe Gln Gly Ser Trp Gly Gln Gln 565 570 575 Leu Ser Met Met LeuTrp Phe Gly Phe Ser Thr Ile Val Phe Ala His 580 585 590 Arg Glu Lys LeuGln Lys Met Ser Ser Arg Phe Leu Val Ile Val Trp 595 600 605 Val Phe ValVal Leu Ile Leu Thr Ser Ser Tyr Ser Ala Asn Leu Thr 610 615 620 Ser ThrLys Thr Ile Ser Arg Met Gln Leu Asn His Gln Met Val Phe 625 630 635 640Gly Gly Ser Thr Thr Ser Met Thr Ala Lys Leu Gly Ser Ile Asn Ala 645 650655 Val Glu Ala Tyr Ala Gln Leu Leu Arg Asp Gly Thr Leu Asn His Val 660665 670 Ile Asn Glu Ile Pro Tyr Leu Ser Ile Leu Ile Gly Asn Tyr Pro Asn675 680 685 Asp Phe Val Met Thr Asp Arg Val Thr Asn Thr Asn Gly Phe GlyPhe 690 695 700 Met Phe Gln Lys Gly Ser Asp Leu Val Pro Lys Val Ser ArgGlu Ile 705 710 715 720 Ala Lys Leu Arg Ser Leu Gly Met Leu Lys Asp MetGlu Lys Lys Trp 725 730 735 Phe Gln Lys Leu Asp Ser Leu Asn Val His SerAsn Thr Glu Glu Val 740 745 750 Ala Ser Thr Asn Asp Asp Asp Glu Ala SerLys Arg Phe Thr Phe Arg 755 760 765 Glu Leu Arg Gly Leu Phe Ile Ile AlaGly Ala Ala His Val Leu Val 770 775 780 Leu Ala Leu His Leu Phe His ThrArg Gln Glu Val Ser Arg Leu Cys 785 790 795 800 Thr Lys Leu Gln Ser PheTyr Lys 805 6 amino acids amino acid single linear protein 34 Lys ValIle His Arg Ser 1 5 3 amino acids amino acid single linear protein 35Ala Leu Leu 1 7 amino acids amino acid single linear peptide 36 Gln LeuThr Gly Gln Asp Ser 1 5

What is claimed is:
 1. A method for identifying a compound thatmodulates the growth of a plant through a glutamate-related process,comprising: a) exposing a plant that expresses or over expresses a plantglutamate receptor to a test compound in the absence of glutamate,wherein the plant glutamate receptor has glutamate binding activity andcomprises an amino acid sequence selected from the following: i) anamino acid sequence encoded by a nucleic acid that hybridizes underhighly stringent conditions to the complement of the coding sequence ofthe nucleotide sequence of SEQ ID NO: 13, 14, or 15, wherein said highlystringent conditions include washing in 65° C. in 0.5×SSC; ii) theglutamate binding domain comprising SEQ ID NO: 1; iii) the amino acidsequence comprising SEQ ID NO: 33; iv) the glutamate binding domaincomprising SEQ ID NO: 8; and v) the glutamate binding domain comprisingSEQ ID NO :12; b) exposing a plant that expresses or over expresses theplant glutamate receptor described in step a) to the test compound inthe presence of glutamate; and c) measuring and comparing growth of theplant of step a) with that of the plant of step b); in which adifference in growth indicates that the compound modulates plant growththrough a glutamate-related process.
 2. A method for identifying acompound that modulates the activity of a plant glutamate receptor,comprising: a) exposing a genetically engineered host cell to a testcompound in the absence of glutamate, wherein the host cell bothexpresses or over expresses a plant glutamate receptor and responds tothe signal transduced by the receptor, and the plant glutamate receptorhas glutamate binding activity and comprises an amino acid sequenceselected from the following: i) an amino acid sequence encoded by anucleic acid that hybridizes under highly stringent conditions to thecomplement of the coding sequence of the nucleotide sequence of SEQ IDNO: 13, 14, or 15, wherein said highly stringent conditions includewashing in 65° C. in 0.5×SSC; ii) the glutamate binding domaincomprising SEQ ID NO: 1; iii) the amino acid sequence comprising SEQ IDNO: 33; iv) the glutamate binding domain comprising SEQ ID NO: 8; and v)the glutamate binding domain comprising SEQ ID NO:12; b) exposing agenetically engineered host cell to the test compound in the presence ofglutamate that expresses or over expresses the plant glutamate receptordescribed in step a); and c) measuring and comparing the response of thehost cell of step a) with that of the host cell of step b); in which adifference in the response of the cells indicates that the compoundmodulates the activity of a plant glutamate receptor.
 3. A method foridentifying a compound that binds to a plant glutamate receptor,comprising: a) exposing a genetically engineered host cell thatexpresses or over expresses the plant glutamate receptor to a testcompound, wherein the plant glutamate receptor comprises an amino acidsequence selected from the following: i) an amino acid sequence encodedby a nucleic acid that hybridizes under highly stringent conditions tothe complement of the coding sequence of the nucleotide sequence of SEQID NO: 13, 14, or 15, wherein said highly stringent conditions includewashing in 65° C. in 0.5×SSC; ii) the glutamate binding domain of SEQ IDNO: 1; iii) the amino acid sequence of SEQ ID NO: 33; iv) the glutamatebinding domain of SEQ ID NO: 8; and v) the glutamate binding domain ofSEQ ID NO:12; and b) measuring the binding of the test compound to theglutamate receptor expressed or over expressed in the host cell, inwhich binding of the test compound to the plant glutamate receptorindicates that the test compound interacts with the plant glutamatereceptor.
 4. A method for identifying a compound that binds to a plantglutamate receptor, comprising: a) exposing an isolated plant glutamatereceptor to the test compound, wherein the plant glutamate receptorcomprises an amino acid sequence selected from the following: i) anamino acid sequence encoded by a nucleic acid that hybridizes underhighly stringent conditions to the complement of the coding sequence ofthe nucleotide sequence of SEQ ID NO: 13, 14, or 15, wherein said highlystringent conditions include washing in 65° C. in 0.5×SSC; ii) theglutamate binding domain of SEQ ID NO: 1; iii) the amino acid sequenceof SEQ ID NO: 33; iv) the glutamate binding domain of SEQ ID NO: 8; andv) the glutamate binding domain of SEQ ID NO:12; and b) measuring thebinding of the test compound to the isolated plant glutamate receptor.5. A method for identifying a compound that modulates the growth of aplant through a glutamate-related process, comprising: a) exposing aplant that expresses or over expresses a plant glutamate receptor to atest compound in the presence of glutamate, wherein the plant glutamatereceptor has glutamate binding activity and comprises an amino acidsequence selected from the following: i) an amino acid sequence encodedby a nucleic acid that hybridizes under highly stringent conditions tothe complement of the coding sequence of the nucleotide sequence of SEQID NO: 13, 14, or 15, wherein said highly stringent conditions includewashing in 65° C. in 0.5×SSC; ii) the glutamate binding domaincomprising SEQ ID NO: 1; iii) the amino acid sequence comprising SEQ IDNO: 33; iv) the glutamate binding domain comprising SEQ ID NO: 8; and v)the glutamate binding domain comprising SEQ ID NO:12; b) exposing aplant that expresses or over expresses the plant glutamate receptordescribed in step a) to the test compound in the presence of bothglutamate and a glutamate-antagonist; and c) measuring and comparinggrowth of the plant of step a) with that of the plant of step b); inwhich a difference in growth indicates that the compound modulates plantgrowth through a glutamate-related process.
 6. A method for identifyinga compound that modulates the activity of a plant glutamate receptor,comprising: a) exposing a genetically engineered host cell to a testcompound in the presence of glutamate, wherein the host cell bothexpresses or over expresses a plant glutamate receptor and responds tothe signal transduced by the receptor, and the plant glutamate receptorhas glutamate binding activity and comprises an amino acid sequenceselected from the following: i) an amino acid sequence encoded by anucleic acid that hybridizes under highly stringent conditions to thecomplement of the coding sequence of the nucleotide sequence of SEQ IDNO: 13, 14, or 15, wherein said highly stringent conditions includewashing in 65° C. in 0.5×SSC; ii) the glutamate binding domaincomprising SEQ ID NO: 1; iii) the amino acid sequence comprising SEQ IDNO: 33; iv) the glutamate binding domain comprising SEQ ID NO: 8; and v)the glutamate binding domain comprising SEQ ID NO:12; b) exposing agenetically engineered host cell to the test compound in the presence ofboth glutamate and a glutamate-antagonist, wherein the host cellexpresses or over expresses the plant glutamate receptor described instep a); and c) measuring and comparing the response of the host cell ofstep a) with that of the host cell of step b); in which a difference inthe response of the cells indicates that the compound modulates theactivity of a plant glutamate receptor.
 7. The method of claim 1 or 5wherein the plant exposed in step a) and the plant exposed in step b)have been altered to effect the sensitivity of the plant to the testcompound.